Method and probes for the detection of chromosome aberrations

• US 20040043383A1
• Filed: 08/27/2002
• Published: 03/04/2004
• Est. Priority Date: 05/04/1998
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Dako A/S (Agilent Technologies, Inc.)
• Inventors: Maria Van Dongen, Jacobus Johannus, Nielsen, Kirsten Vang, Adelhorst, Kim, Pluzek, Karl-Johan
• Granted Patent: US 7,105,294 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 in a sample of eukaryotic origin using in situ hybridisation, comprising the steps of

a) producing a preparation of the sample, whereby the sample will be subject to a fixation,

b) contacting said preparation with a hybridisation solution comprising 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 one or more peptide nucleic acid probes capable of hybridising to specific nucleic acid sequences related to another or the same potential aberration in a chromosome, 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,

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

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

2. A method according to claim 1, wherein each set of hybridisation probes hybridise to specific nucleic acid sequences related to the same potential chromosome aberration.

3. A method according to claim 1 or 2, wherein the potential aberration is a potential breakpoint.

7. A method according to any one of claims 1 to 6 wherein each peptide nucleic acid probe contains from 8 to 40 polymerised moieties.

8. A method according to any one of claims 1 to 7 wherein the peptide nucleic acid probe comprises polymerised moieties of formula (I)

9. A method according to claim 8, wherein Y, X, Q, u, p, q, r, s, R₂, R₅, R₇and R₈ are as defined in claim 8, t is 0, R₁ designates H or CH_3, and each of R₃, R₄, R₆ and R₉ designates H.

10. A method according to claim 8, 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,

11. A method according to claim 8, 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

12. A method according to claim 8, 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,

13. A method according to claim 12, 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.

14. A method according to claim 8, 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:

15. A method according to any one of claims 12 to 14, wherein polymerised moieties of formulas (IV)-(VII) constitute at least 75% by weight of the peptide nucleic acid probe.

16. A method according to any one of claims 1 to 15, wherein the hybrid destabilising agent is selected from the group consisting of formamide, urea, guanidine, ethylene glycol, and glycerol.

17. A method according to any one of claims 1 to 16, wherein the concentration of the hybrid destabilising agent is above 10%.

18. A method according to any one of claims 1 to 17 wherein each peptide nucleic acid probe is labelled directly or indirectly with at least one detectable label.

19. A method according to claim 18 wherein the label is selected from the group consisting of enzymes, chromophores, fluorochromes, haptens, dyes and spin labels.

20. A method according to claim 19 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.

21. A method according to any one of claims 18 to 20 wherein the detectable label of one set of hybridisation probes is different from the detectable label of at least one other set of hybridisation probes.

22. A method according to any one of claims 1 to 21, wherein two sets of hybridisation probes are used.

23. A method according to any one of claims 1 to 21, 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. A method according to any one of claims 1 to 23, wherein the sample is a tissue section, a cell smear, a cell suspension, or a chromosome spread.

4. A method of determining the presence of chromosome aberrations in a sample of eukaryotic origin using in situ hybridisation, comprising the steps of

a) producing a preparation of the sample, whereby the sample will be subject to a fixation,

b) contacting said preparation with a hybridisation solution comprising 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 flanking one side of a potential breakpoint in a chromosome, and at least one set comprising one or more peptide nucleic acid probes capable of hybridising to specific nucleic acid sequences flanking the other side of the potential breakpoint 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,

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

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

6. A method according to claim 4 or 5, wherein each set of hybridisation probes hybridise to said specific nucleic acid sequences flanking a potential breakpoint in a chromosome within a genomic distance of no more than 100 kb, preferably no more than 50 kb from the potential breakpoint.

5. A method of determining the presence of chromosome aberrations in a sample of eukaryotic origin using in situ hybridisation, comprising the steps of

a) producing a preparation of the sample, whereby the sample will be subject to a fixation,

b) contacting said preparation with a hybridisation solution comprising at least two sets of hybridisation probes, one set comprising one or more peptide nucleic acid probes capable of hybridising to specific nucleic acid sequences flanking one side of a potential breakpoint in a chromosome, and one set comprising one or more peptide nucleic acid probes capable of hybridising to specific nucleic acid sequences flanking the other side of the potential breakpoint 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,

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

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

25. A pair of sets of hybridisation probes, one set hybridising to specific nucleic acid sequences related to a potential aberration of a chromosome, and one set hybridising to specific nucleic acid sequences related to another or the same potential aberration of a chromosome, each set comprising one or more peptide nucleic acid probes of formula (I) to (VII).

26. A pair of sets of hybridisation probes according to claim 25, each set capable of hybridising to nucleic acid sequences related to different potential chromosome aberrations.

27. A pair of sets of hybridisation probes according to claim 25 or 26, each set of hybridisation probes hybridising to said specific nucleic acid sequences related to the same potential aberration.

28. A pair of sets of hybridisation probes according to claim 27, each set of probes hybridising to nucleic acid sequences related to a potential breakpoint.

31. A pair of sets of hybridisation probes according to claim 25 to 30 characterised in that each set comprises from 1 to 500, from 1 to 250, from 1 to 100, from 1 to 35 peptide nucleic acid probes.

29. A pair of sets of hybridisation probes, one set hybridising to specific nucleic acid sequences flanking one side of a potential breakpoint in a chromosome and another set hybridising to specific nucleic acid sequences flanking the other side of the potential breakpoint and each set comprising one or more peptide nucleic acid probes of formula (I) to (VII).

30. A pair of sets of hybridisation probes according to claim 29, each set hybridising to specific nucleic acid sequences flanking either side of a potential breakpoint in a chromosome within a genomic distance of no more than 100 kb, preferably no more than 50 kb from the potential breakpoint.

32. An in situ diagnostic method for diagnosing chromosome aberrations in a sample of eukaryotic origin comprising the steps of

a) producing a preparation of the sample, whereby the sample will be subject to a fixation,

b) contacting said preparation with a hybridisation solution comprising 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 one or more peptide nucleic acid probes capable of hybridising to specific nucleic acid sequences related to another potential aberration in a chromosome, 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,

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

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

33. An in situ diagnostic method for diagnosing chromosome aberrations in a sample of eukaryotic origin comprising the steps of

a) producing a preparation of the sample, whereby the sample will be subject to a fixation,

b) contacting said preparation with a hybridisation solution comprising 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 flanking one side of a potential breakpoint in a chromosome, and at least one set comprising one or more peptide nucleic acid probes capable of hybridising to specific nucleic acid sequences flanking the other side of the potential breakpoint 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,

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

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

34. An in situ diagnostic method for diagnosing chromosome aberrations in a sample of eukaryotic origin comprising the steps of

a) producing a preparation of the sample, whereby the sample will be subject to a fixation,

b) contacting said preparation with a hybridisation solution comprising at least two sets of hybridisation probes, one set comprising one or more peptide nucleic acid probes capable of hybridising to specific nucleic acid sequences flanking one side of a potential breakpoint in a chromosome, and one set comprising one or more peptide nucleic acid probes capable of hybridising to specific nucleic acid sequences flanking the other side of the potential breakpoint 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,

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

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