# US 2014/0194316 A1 · Enhanced multiplex fish

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- Number: US 2014/0194316 A1
- Document ID: US20140194316A1
- Status: Published application
- Kind: A1
- Application number: 14/151,111
- Filed: 2014-01-09
- Published: 2014-07-10
- Earliest claimed: 2010-03-04
- Assignees: Miacom Diagnostics GmbH
- Applicants: MIACOM DIAGNOSTICS GmbH
- Inventors: Ian Peter THRIPPLETON; Walter FREIHERR VON STEIN
- Web page: https://patentgrep.com/patent/US20140194316A1

## CPC

- C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence)
- C12Q 1/6841: In situ hybridisation
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

Subject of the present invention is a combination of nucleic acid molecules capable of hybridizing 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

### Claim 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.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 1)

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

### Claim 4 (depends on 1)

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.

### Claim 5 (depends on 1)

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.

### Claim 6 (depends on 1)

The combination according to claim 1, wherein the nucleic acid molecules of the combination independently hybridize with the target sequence
(i) with a txG in the range of −15 to −25 kcal/mol,
(ii) with a combined ΔG 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 ΔG than the ΔG generated by the natural refolding of the target sequence.

### Claim 7

(canceled)

### Claim 8

(canceled)

### Claim 9 (depends on 1)

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

### Claim 10 (depends on 1)

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.

### Claim 11 (depends on 10)

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

### Claim 12 (depends on 10)

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

### Claim 13 (depends on 10)

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

### Claim 14 (depends on 10)

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

### Claim 15 (depends on 1)

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

### Claim 16 (depends on 2)

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

### Claim 17 (depends on 3)

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

### Claim 18 (depends on 5)

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

### Claim 19 (depends on 10)

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

### Claim 20 (depends on 12)

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

### Claim 21 (depends on 1)

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.

### Claim 22 (depends on 1)

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.