SET OF OLIGONUCLEOTIDE PROBES AS WELL AS METHODS AND USES THERETO

• US 20160230221A1
• Filed: 04/22/2016
• Published: 08/11/2016
• 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
• Granted Patent: US 10,640,815 B2
• Time in Patent Office: Days
• 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 method of detecting a genomic target sequence of interest in a sample, said method comprising the steps of:

incubating a set of oligonucleotide probes 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 by one of a chromogenic reaction and a metallographic reaction,

wherein the set of oligonucleotide probes comprises 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 squally spaced positions, and

wherein the detecting step comprises one of a chromogenic reaction and a metallographic reaction,

2. The method according to claim 1, wherein the incubating step and the detecting step include a chromogenic in situ hybridization assay.

3. The method according to claim 1, wherein the genomic target sequence of interest is a region spread over different regions of one chromosome or spread over different chromosomes.

4. The method according to claim 1, wherein the time for performing the incubating step is less than 91 minutes.

5. The method according to claim 1, wherein the at least three labels are incorporated into the oligonucleotide probes with a phosphoramidite modified building block.

6. The method according to claim 1, wherein the at least three labels are incorporated into the oligonucleotide probes via an oxidation step of phosphoramidite based oligonucleotide synthesis.

7. The method according to claim 1, wherein the at least three labels include one of a hapten, a radioisotope, biotin, digoxigenin, or a 2,4-dinitrophenyl moiety.

8. The method according to claim 1, wherein the at least three labels in each of the oligonucleotide probes are positioned at locations separated by at least 16 nucleotides.

9. A method of detecting a genomic target sequence of interest in a sample, wherein the genomic target sequence of interest is a region spread over different chromosomes, said method comprising the steps of:

incubating a set of oligonucleotide probes with a sample comprising the 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,

wherein the set of oligonucleotide probes comprises 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 at least 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.

10. The method according to claim 9, wherein the incubating step and the detecting step include an in situ hybridization assay.

11. The method according to claim 10, wherein the in situ hybridization assay is chromogenic.

12. The method according to claim 9, wherein the detecting step comprises one of a chromogenic reaction and a metallographic reaction.

13. The method of claim 9, wherein the at least three labels in each of the oligonucleotide probes are positioned at locations separated by of at least 16 nucleotides.

14. The method of claim 9, wherein the at least three labels include one of a hapten, a radioisotope, biotin, digoxigenin or a 2,4-dinitrophenyl moiety.

15. 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 a detection agent specific for the detection of hybridization of at least one of the oligonucleotide probes to at least one of the subsequences of the genomic target of interest in a chromogenic reaction or a metallographic reaction.

16. The kit of claim 15, comprising a detection agent specific for the detection of hybridization of at least one of the oligonucleotide probes to at least one of the subsequences of the genomic target of interest in an in situ hybridization assay.

17. The kit of claim 16, wherein the in situ hybridization assay is chromogenic.

18. The kit of claim 15, wherein the label is detected by a chromogenic reaction or a metallographic reaction.

19. A kit for the detection of a genomic target sequence of interest, wherein the genomic target sequence of interest is a region which is spread over different regions of one chromosome or spread over different chromosomes, 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, each of the oligonucleotide probes comprising at least three labels, each of the at least 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;

and a detection agent.

20. The kit of claim 19, wherein said detection agent is specific for the detection of hybridization of at least one of the oligonucleotide probes to at least one of the subsequences of the genomic target of interest in a chromogenic reaction or a metallographic reaction.

21. The kit of claim 19, wherein the label is detected by a chromogenic reaction or a metallographic reaction.

22. A set of oligonucleotide probes for detecting a genomic target of interest, the set comprising at least 100 oligonucleotide probes, the oligonucleotide probes each 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 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 the at least three labels are detectable by one of a chromogenic reaction and a metallographic reaction.

23. The set of claim 22, wherein the at least three labels include one of a hapten, a radioisotope, biotin, digoxigenin or a 2,4-dinitrophenyl moiety.

24. The set of claim 22, wherein the at least three labels in each of the oligonucleotide probes are positioned at locations separated by at least 16 nucleotides in each of the oligonucleotide probes.

25. The set of claim 22, wherein said at least 100 oligonucleotide probes comprise one of at least 200, 300, 400, 500, and 1,000 different oligonucleotide probes.

26. The set of claim 22, wherein each of the oligonucleotide probes comprise a length of at least 20 to not greater than 200 nucleotides.

27. The set of claim 22, 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.

28. The set of claim 22, comprising at least 1000 labels.

30. The set of claim 22, wherein the oligonucleotide probes are clustered within the genomic target sequence of interest.

31. A set of oligonucleotide probes for detecting a genomic target of interest, wherein the genomic target sequence of interest is a region spread over different regions of one chromosome or spread over different chromosomes, 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.