Set of oligonucleotide probes as well as methods and uses thereto

• US 9,347,091 B2
• Filed: 04/15/2013
• Issued: 05/24/2016
• Est. Priority Date: 07/30/2009
• Status: Active Grant

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Roche Molecular Systems (Roche Holding AG)
• Inventors: Bergmann, Frank, Eberle, Walter, Fischer, Thomas, von der Eltz, Herbert
• Primary Examiner(s): Lu, Frank
• Application Number:
  US13/862,530
• Publication Number:
  US 20130203628A1
• Time in Patent Office: 1,135 Days
• Field of Search: 435/6.1, 435/6.11, 435/91.1, 435/183, 435/283.1, 435/287.1, 435/287.2, 436/94, 436/501, 536/23.1, 536/24.3, 536/24.33
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/1025 labels being on the same oligonucleotide

Abstract

The present disclosure relates to a set of at least 100 single-stranded oligonucleotide probes directed against (or complementary to) portions of a genomic target sequence of interest. The present disclosure also relates to a method of detecting a genomic target sequence of interest using the set of oligonucleotide probes and a method of generating the set of oligonucleotide probes. Further, the present disclosure relates to a kit comprising the set of oligonucleotide probes and at least one further component.

Claims

1. A set of oligonucleotide probes for detecting a genomic target sequence of interest, the set comprising at least 100 oligonucleotide probes, each of the oligonucleotide probes comprising a different nucleotide sequence complementary to a different subsequence of the genomic target sequence of interest, each of the oligonucleotide probes comprising at least three labels, each of the three labels attached to one of a base, a sugar, and a phosphate moiety of a nucleotide in each of the oligonucleotide probes at about equally spaced positions, wherein each of the oligonucleotide probes has an identical length, and the at least three labels are positioned at identical positions in each of the oligonucleotide probes.

2. The set of claim 1, wherein said at least 100 oligonucleotide probes comprises one of at least 200, 300, 400, 500, and 1,000 different oligonucleotide probes.

3. The set of claim 1, wherein each of the oligonucleotide probes comprise a length of at least 20 to not greater than 200 nucleotides.

4. The set of claim 1, wherein the at least three labels of each of the oligonucleotide probes are positioned at locations separated by 10 to 20 nucleotides.

5. The set of claim 1, wherein the at least three labels are attached to the one of the base, the sugar, and the phosphate moiety of the nucleotide indirectly via a linker.

6. The set of claim 1, wherein the at least three labels are detectable by one of a chromogenic reaction and a metallographic reaction.

7. The set of claim 1, wherein the at least three labels include one of a fluorescein dye, a rhodamine dye, a cyanine dye, a biotin, a digoxigenin, and a 2,4-dinitrophenyl moiety.

8. The set of claim 1 comprising at least 1000 labels.

9. The set of claim 1, wherein the oligonucleotide probes are clustered within the genomic target sequence of interest.

10. The set of claim 1, wherein each of the oligonucleotide probe further comprises a non-nucleotide unit positioned at an identical location in each of the oligonucleotide probes.

11. A method of detecting a genomic target sequence of interest in a sample, said method comprising the steps of:

incubating the set of oligonucleotide probes in claim 1 with a sample comprising a genomic target sequence of interest under conditions for the binding of the set of oligonucleotide probes to the genomic target sequence of interest; and

detecting hybridizations of a plurality of the oligonucleotide probes of the set with the target sequence of interest, thereby detecting the genomic target sequence of interest in the sample.

12. The method according to claim 11, wherein the detecting step comprises one of a chromogenic reaction and a metallographic reaction.

13. The method according to claim 11, wherein the time for performing the incubating step is less than 91 minutes.

14. The method according to claim 11, wherein the incubating step and the detecting step includes in an in situ hybridization assay.

15. The method of claim 11, wherein each of the oligonucleotide probe further comprises a non-nucleotide unit positioned at an identical location in each of the oligonucleotide probes.

16. A kit for the detection of a genomic target sequence of interest, the kit comprising:

a set of at least 100 oligonucleotide probes, each of the oligonucleotide probes having at least three labels and comprising a different nucleotide sequence complementary to a different subsequence of the genomic target sequence of interest, wherein each of the at least three labels is attached to one of a base, a sugar, and a phosphate moiety of a nucleotide in each of the oligonucleotide probes at about equally spaced positions and wherein each of the oligonucleotide probes has an identical length, and the at least three labels are positioned at identical locations in each of the oligonucleotide probes; and

a detection agent.

17. The kit of claim 16, wherein the detection agent is specific for detecting the hybridization of at least one of the oligonucleotide probes to at least one of the subsequences of the genomic target sequence of interest in an in situ hybridization assay.

18. The kit of claim 16, wherein each of the oligonucleotide probe further comprises a non-nucleotide unit positioned at an identical location in each of the oligonucleotide probes.

19. The kit of claim 16, wherein the at least three labels of each of the oligonucleotide probes are positioned at locations separated by 10 to 20 nucleotides.