Method and probes for the detection of chromosome aberrations

• US 7,368,245 B2
• Filed: 09/08/2005
• Issued: 05/06/2008
• Est. Priority Date: 05/04/1998
• Status: Expired due to Fees

• Current Assignee: Dako Denmark A/S (Agilent Technologies, Inc.)
• Original Assignee: Dako Denmark A/S (Agilent Technologies, Inc.)
• Inventors: Van Dongen, Jacobus Johannus Maria, Nielsen, Kirsten Vang, Adelhorst, Kim, Pluzek, Karl-Johan
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US11/220,718
• Publication Number:
  US 20060160106A1
• Time in Patent Office: 971 Days
• Field of Search: 536/23.1, 536/24.3, 536/24.33, 435/6
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2525/107 incorporating a peptide nucleic acid
• CPC Class Codes: C12Q 2565/601 being a microscope, e.g. atomic force microscopy [AFM]
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

A novel method for detecting chromosome aberrations is disclosed. More specifically, chromosome aberrations are detected by in situ hybridisation using at least two sets of hybridisation probes, at least one set comprising one or more peptide nucleic acid probes capable of hybridising to specific nucleic acid sequences related to a potential aberration in a chromosome, and at least one set comprising two or more peptide nucleic acid probes capable of hybridising to specific nucleic acid sequences related to another potential aberration in a chromosome. In particular, the method may be used for detecting chromosome aberrations in the form of breakpoints.

Claims

1. A method of determining the presence of chromosome aberrations, wherein said aberrations are selected from deletions, amplifications, inversions or aneuploidy in a chromosome in a sample of eukaryotic origin, using in situ hybridisation, comprising the steps of

a) contacting said sample with a hybridisation solution comprising at least two sets of hybridisation probes, wherein at least one set comprises one or more probes capable of hybridising to specific nucleic acid sequences related to a potential aberration in a chromosome, and at least one set comprises one or more probes capable of hybridising to specific nucleic acid sequences related to another or the same potential aberration in a chromosome, wherein at least one set of hybridisation probes is a set of peptide nucleic acid probes, and wherein each probe is labelled directly or indirectly with at least one detectable label, wherein the detectable label of the at least one set of hybridisation probes is different from the detectable label of the at least one other set of hybridisation probes and optionally a hybrid destabilising agent in an amount effective to decrease the melting temperature of hybrids formed between said nucleic acid sequences and said peptide nucleic acid probes so as to increase the ratio between specific and non-specific binding,

b) removing unbound and non-specifically bound peptide nucleic acid probe, and

c) determining the presence of the bound peptide nucleic acid probe in the preparation.

2. The method of claim 1, wherein each set of hybridisation probes hybridises to specific nucleic acid sequence related to the same potential chromosome aberration.

3. The method of claim 1, wherein each set of hybridisation probes hybridises to specific nucleic acid sequences related to different potential chromosome aberrations.

4. The method of claim 1, wherein the at least one set of hybridisation probes hybridise to said specific nucleic acid sequences flanking a potential aberration in a chromosome.

5. The method of claim 1, wherein the method comprises a step of producing a preparation of the sample, whereby the sample will be subject to a fixation, wherein said step is preceding step a).

6. The method of claim 1, wherein each peptide nucleic acid probe contains from 8 to 40 polymerised moieties.

7. The method of claim 1, wherein the peptide nucleic acid probe comprises polymerised moieties of formula (I)

8. The method of claim 7, wherein Y, X, Q, u, p, q, r, s, R₂, R₅, R₇ and R₈ are as defined in claim 7, t is 0, R₁ designates H or CH₃, and each of R₃, R₄, R₆ and R₉ designates H.

9. The method of claim 7, wherein the peptide nucleic acid probe comprises polymerised moieties of formula (II), which are moieties of the general formula (I) wherein r is 0 and q and s are 1,

10. The method of claim 7, wherein the peptide nucleic acid probe comprises polymerised moieties of formula (III), which are moieties of the general formula (I) wherein p, r and t are 0, and q and s are 1

11. A method of claim 7, wherein the peptide nucleic acid probe comprises polymerised moieties selected among those of formulas (IV)-(VI), which are moieties of the general formula (I) wherein p, r and t are 0, and u, s and q are 1,

12. The method of claim 10, wherein R₂, R₅ and R₇ designate H or the side chain of Ala, Asp, Cys, Glu, His, HomoCys, Lys, Orn, Ser or Thr, and Q designates thymine, adenine, cytosine, guanine, uracil or hypoxanthine.

13. The method of claim 9, wherein the peptide nucleic acid probe comprises polymerised moieties of formula (VII), which are moieties of formula (I) wherein p, r and t are 0, and u, s and q are 1:

14. The method of claim 10, wherein polymerised moieties of formulas (IV)-(VII) constitute at least 75% by weight of the peptide nucleic acid probe.

15. The method of claim 11, wherein polymerised moieties of formulas (IV)-(VII) constitute at least 75% by weight of the peptide nucleic acid probe.

16. The method of claim 12, wherein polymerised moieties of formulas (IV)-(VII) constitute at least 75% by weight of the peptide nucleic acid probe.

17. The method of claim 13, wherein polymerised moieties of formulas (IV)-(VII) constitute at least 75% by weight of the peptide nucleic acid probe.

18. The method of claim 1, wherein the hybrid destabilising agent is selected from the group consisting of formamide, urea, guanidine, ethylene glycol, and glycerol.

19. The method of claim 1, wherein the concentration of the hybrid destabilising agent is above 10%.

20. The method of claim 1 wherein the label is selected from the group consisting of enzymes, chromophores, fluorochromes, haptens, dyes and spin labels.

21. The method of claim 20 wherein the label is selected from the group consisting of fluorescein labels, biotin, digoxigenin, dinitro benzoic acid, peptide labels, rhodamine, R-phycoerythrine and cyanine dyes.

22. The method of claim 1, wherein two sets of hybridisation probes are used.

23. The method of claim 1, wherein each set of hybridisation probes comprises from 1 to 500, from 1 to 250, from 1 to 100, or from 1 to 35 peptide nucleic acid probes.

24. The method of claim 1, wherein the sample is a tissue section, a cell smear, a cell suspension, or a chromosome spread.

41. The method of claim 7, wherein the hybrid destabilising agent is selected from the group consisting of formamide, urea, guanidine, ethylene glycol, and glycerol.

42. The method of claim 7, wherein the concentration of the hybrid destabilising agent is above 10%.

43. The method of claim 7, wherein the label is selected from the group consisting of enzymes, chromophores, fluorochromes, haptens, dyes and spin labels.

44. The method of claim 43, wherein the label is selected from the group consisting of fluorescein labels, biotin, digoxigenin, dinitro benzoic acid, peptide labels, rhodamine, R-phycoerythrine and cyanine dyes.

25. A method of determining the presence of a gene amplification in a sample of eukaryotic origin, using in situ hybridisation, comprising the steps of

a) contacting said sample with a hybridisation solution comprising at least two sets of hybridisation probes, wherein at least one set comprises one or more peptide nucleic acid probes capable of hybridising to specific nucleic acid sequences in a chromosome, and at least one set comprises one or more probes capable of hybridising to specific nucleic acid sequences related to the gene amplification, wherein each probe is labelled directly or indirectly with at least one detectable label, wherein the detectable label of the at least one set of hybridisation probes is different from the detectable label of the at least one other set of hybridisation probes and optionally a hybrid destabilising agent in an amount effective to decrease the melting temperature of hybrids formed between said nucleic acid sequences and said peptide nucleic acid probes so as to increase the ratio between specific and non-specific binding,

b) removing unbound and non-specifically bound probes, and

c) determining the presence of the bound probes in the preparation.

26. The method of claim 25, wherein at least one set of hybridisation probes comprises one or more probes capable of hybridising to the HER-2 gene.

27. The method of claim 26, wherein the one or more peptide nucleic acid probes are capable of hybridizing to a centromere.

28. The method of claim 27, wherein the method comprises a step of producing a preparation of the sample, whereby the sample will be subject to a fixation, wherein said step is preceding step a).

29. The method of claim 27, wherein each peptide nucleic acid probe contains from 8 to 40 polymerised moieties.

30. The method of claim 27, wherein the peptide nucleic acid probe comprises polymerised moieties of formula (I)

31. The method of claim 30, wherein Y, X, Q, u, p, q, r, s, R₂, R₅, R₇ and R₈ are as defined in claim 30, t is 0, R₁ designates H or CH₃, and each of R₃, R₄, R₆ and R₉ designates H.

32. The method of claim 30, wherein the peptide nucleic acid probe comprises polymerised moieties of formula (II), which are moieties of the general formula (I) wherein r is 0 and q and s are 1,

33. The method of claim 30, wherein the peptide nucleic acid probe comprises polymerised moieties of formula (III), which are moieties of the general formula (I) wherein p, r and t are 0, and q and s are 1

34. A method of claim 30, wherein the peptide nucleic acid probe comprises polymerised moieties selected among those of formulas (IV)-(VI), which are moieties of the general formula (I) wherein p, r and t are 0, and u, s and q are 1,

35. The method of claim 33, wherein R₂, R₅ and R₇ designate H or the side chain of Ala, Asp, Cys, Glu, His, HomoCys, Lys, Orn, Ser or Thr, and Q designates thymine, adenine, cytosine, guanine, uracil or hypoxanthine.

36. The method of claim 32, wherein the peptide nucleic acid probe comprises polymerised moieties of formula (VII), which are moieties of formula (I) wherein p, r and t are 0, and u, s and q are 1:

37. The method of claim 33, wherein polymerised moieties of formulas (IV)-(VII) constitute at least 75% by weight of the peptide nucleic acid probe.

38. The method of claim 34, wherein polymerised moieties of formulas (IV)-(VII) constitute at least 75% by weight of the peptide nucleic acid probe.

39. The method of claim 35, wherein polymerised moieties of formulas (IV)-(VII) constitute at least 75% by weight of the peptide nucleic acid probe.

40. The method of claim 36, wherein polymerised moieties of formulas (IV)-(VII) constitute at least 75% by weight of the peptide nucleic acid probe.

45. The method of claim 27, wherein two sets of hybridisation probes are used.

46. The method of claim 27, wherein each set of hybridisation probes comprises from 1 to 500, from 1 to 250, from 1 to 100, or from 1 to 35 peptide nucleic acid probes.

47. The method of claim 27, wherein the sample is a tissue section, a cell smear, a cell suspension, or a chromosome spread.