Method and probes for the detection of chromosome aberrations

• US 7,642,057 B2
• Filed: 02/19/2008
• Issued: 01/05/2010
• 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: Adelhorst, Kim, Pluzek, Karl-Johan, Van Dongen, Jacobus Johannus Maria, Nielsen, Kristen Vang
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US12/071,268
• Publication Number:
  US 20080187934A1
• Time in Patent Office: 686 Days
• Field of Search: 435/6, 536/23.1, 536/24, 536/3, 536/24.33, 536/25.3
• US Class Current: 435/6.12
• 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 a gene deletion in a sample of eukaryotic origin, using in situ hybridization, comprising the steps of:

a) contacting said sample with a hybridization solution comprising at least two sets of hybridization 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 deletion, 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 hybridization probes is different from the detectable label of the at least one other set of hybridization 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.

2. The method of claim 1, wherein at least one set of hybridization probes comprises one or more probes capable of hybridising to the p53, RB, or TALL genes.

3. The method of claim 1, wherein the one or more peptide nucleic acid probes are capable of hybridizing to a centromere.

4. 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).

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

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

7. The method of claim 6, wherein Y, X, Q, u, p, q, r, s, R₂, R₅, R₇ and R₈ are as defined in claim 6, t is 0, R₁ designates H or CH₃, and each of R₃, R₄, R₆ and R₉ designates H.

8. The method of claim 6, wherein the peptide nucleic acid probe comprises polymerised moieties of formula (II),

9. The method of claim 6, wherein the peptide nucleic acid probe comprises polymerised moieties of formula (III),

10. A method of claim 6, wherein the peptide nucleic acid probe comprises polymerised moieties selected among those of formulas (IV)-(VI),

11. The method of claim 9, wherein R₂, R₅ and R₇ designate H or the side chain of Ala, Asp, Cys, Glu, H is, HomoCys, Lys, Orn, Ser or Thr, and Q designates thymine, adenine, cytosine, guanine, uracil or hypoxanthine.

12. The method of claim 8, wherein the peptide nucleic acid probe comprises polymerised moieties of formula (VII),

13. The method of claim 9, wherein polymerised moieties of formulas (IV)-(VII) constitute at least 75% by weight of the peptide nucleic acid probe.

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

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

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

20. The method of 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. The method of claim 1, wherein two sets of hybridization probes are used.

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

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