ENHANCED MULTIPLEX FISH

• US 20130040847A1
• Filed: 03/04/2011
• Published: 02/14/2013
• Est. Priority Date: 03/04/2010
• Status: Abandoned Application

• Current Assignee: MetaSystems Indigo GmbH
• Original Assignee: Miacom Diagnostics GmbH (MetaSystems Indigo GmbH)
• Inventors: Thrippleton, Ian Peter, Freiherr Von Stein, Walter
• Application Number:
  US13/582,651
• Publication Number:
  US 20130040847A1
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 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

Abstract

Subject of the present invention is a combination of nucleic acid molecules capable of hybridising with a target nucleic acid sequence. In order to overcome problems with the reproducibility of FISH assays and to decrease assay time, hairpin probes are used in combination with helper probes annealing adjacent to the target site of the hairpin probe.

Claims

1. A combination of nucleic acid molecules capable of hybridizing with a target nucleic acid sequence, wherein the combination comprises

(a) at least one first nucleic acid molecule comprising

(i) a sequence capable of hybridizing with the target sequence,

(ii) two complementary sequences capable of forming a stem, and

(iii) a luminescent group and a quencher group, wherein the quencher group quenches the fluorescent group if the nucleic acid forms a stem-loop structure, and wherein the fluorescent group is capable of emitting a luminescence signal upon excitation if the oligonucleotide is hybridized with the target sequence,

(b) a second nucleic acid molecule, a third nucleic acid molecule, and optionally at least one further nucleic acid molecule, wherein the second nucleic acid molecule, the third nucleic acid molecule, and the optional at least one further nucleic acid molecule hybridize with the target sequence at a sequence located 5′

or/and 3′

from the sequence to which the first nucleic acid hybridizes.

2. The combination according to claim 1, wherein the nucleic acids are suitable for in situ hybridization.

3. The combination according to claim 1, wherein the target nucleic acid sequence is selected from DNA sequences and RNA sequences.

4. The combination according to claim 1, wherein the nucleic acid molecules hybridize with the target sequence at locations directly adjacent to each other, or wherein at least two nucleic acid molecules hybridize with the target sequence at locations separated from each other by a gap of at least one nucleotide.

5. The combination according to claim 1, wherein at least one sequence of the nucleic acid molecules hybridizing with the target sequence has a length in the range of 16 to 26 nucleotides.

6. The combination according to claim 1, wherein the nucleic acid molecules of the combination independently hybridize with the target sequence

(i) with a t×

G in the range of −

15 to −

25 kcal/mol,

(ii) with a combined AG in the range of −

60 to −

150 kcal/mol, −

8 to −

150 kcal/mol, or −

100 to −

120 kcal/mol, or/and

(iii) with a more negative AG than the AG generated by the natural refolding of the target sequence.

9. Kit or chip comprising the combination of claim 1 in one container and instruction of how to use the combination in another.

10. A method of identifying a cell, diagnosing the presence of a cell and/or a target sequence in a cell in a sample, comprising

(a) providing a sample,

(b) contacting the sample of (a) with the combination of nucleic acid molecules of claim 1 under conditions allowing hybridization of the oligonucleotides with the target sequences in the cell, and

(c) determining the luminescence of the luminescent group of the first nucleic acid molecule,

wherein the fluorescence of the first oligonucleotide indicates the presence of the target sequence.

11. The method according to claim 10, wherein the sample is selected from biological samples including clinical samples.

12. The method according to claim 10, wherein (b) and (c) are performed in situ.

13. The method according to claim 10, wherein the hybridization buffer employed in step (b) does not contain divalent cations.

14. The method of claim 10, wherein in (b) the composition provided is part of a kit or is on a chip.

15. The combination of claim 1, wherein the combination is contained in of a pharmaceutical composition for diagnosing presence of a cell.

16. The combination according to claim 2, wherein fluorescence in-situ hybridization (FISH) is employed.

17. The combination according to claim 3, wherein the target nucleic acid sequence is a rRNA sequence or a mRNA sequence.

18. The combination according to claim 5, wherein the nucleic acid molecules of the combination independently hybridize with the target sequence

a. with a t×

G in the range of −

15 to −

25 kcal/mol,

b. with a combined AG in the range of −

60 to −

150 kcal/mol, −

8 to −

150 kcal/mol, or −

100 to −

120 kcal/mol, or/and

c. with a more negative AG than the AG generated by the natural refolding of the target sequence.

19. The method of claim 10, wherein said target sequence is associated with antibiotic resistance, toxin production, or/and oncogene expression.

20. The method according to claim 12, wherein (b) and (c) are performed by fluorescence in-situ hybridization (FISH).

21. The combination of nucleic acid molecules according to claim 1, wherein the sequence in (a) (i) hybridizes with the target sequence, the two complimentary sequences in (a) (ii) form a stem and the fluorescent group in (a) (iii) emits a luminescence signal upon excitation if the oligonucleotide is hybridized with the target sequence.

22. The combination of claim 1, wherein the combination comprises in (b) said at least one further nucleic acid molecule and said further nucleic acid molecule hybridizes with the target sequence at said sequence located 5′

or/and 3′

from the sequence to which the first nucleic acid hybridizes.

7. (canceled)

8. (canceled)