Set of oligonucleotide probes as well as methods and uses thereto

• US 8,445,206 B2
• Filed: 01/30/2012
• Issued: 05/21/2013
• Est. Priority Date: 07/30/2009
• Status: Active Grant

• Current Assignee: Roche Diagnostics Operations Incorporated (Roche Holding AG)
• Original Assignee: Roche Diagnostics Operations Incorporated (Roche Holding AG)
• Inventors: Bergmann, Frank, Eberle, Walter, von der Eltz, Herbert, Fischer, Thomas
• Primary Examiner(s): Lu, Frank W
• Application Number:
  US13/361,549
• Publication Number:
  US 20120164643A1
• Time in Patent Office: 477 Days
• Field of Search: 435/6.1, 435/6.11, 435/6.12, 435/183, 435/91.1, 435/91.2, 436/94, 436/501, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 435/6.1
• 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 of interest, the set comprising at least 100 oligonucleotide probes, each of the oligonucleotide probes having an identical length and comprising a different nucleotide sequence complementary to a different subsequence of the genomic target sequence of interest, each of the oligonucleotide probes further comprising a non-nucleotide unit and at least one label, wherein the non-nucleotide unit is positioned at a corresponding identical location of each of the oligonucleotide probes.

2. The set of claim 1, wherein the at least 100 oligonucleotide probes comprise 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 comprises a length of at least 20 to not greater than 200 nucleotides, and wherein each of the oligonucleotide probes further comprises at least two non-nucleotide units, each of the at least two non-nucleotide units being positioned at a corresponding identical location of each of the oligonucleotide probes and having a label attached thereto.

4. The set of claim 3, wherein the at least two non-nucleotide units are positioned at locations separated by 10 to 20 nucleotides.

5. The set of claim 1, wherein the label is attached to one of a base, a sugar, and a phosphate moiety of a nucleotide of each of the oligonucleotide probes.

6. The set of claim 5, wherein the label is attached to the one of the base, the sugar, and the phosphate moiety of the nucleotide indirectly via a linker.

7. The set of claim 1, wherein the at least one label is attached to the non-nucleotide unit.

8. The set of claim 1, wherein the label is detectable by one of a chromogenic reaction and a metallographic reaction when the label is suitable for detection by the chromogenic reaction or the metallographic reaction.

9. The set of claim 1, wherein the label comprises one of a fluorescein dye, a rhodamine dye, a cyanine dye, a biotin, a digoxigenin, and a 2,4-dinitrophenyl moiety.

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

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

12. The set of claim 1, wherein the set further comprises:

a first subset of oligonucleotide probes comprising at least 100 oligonucleotide probes for detecting a first genomic target of interest, each probe within the first subset having a non-nucleotide unit, a label, and an identical length, and each probe within the first subset further comprising a different nucleotide sequence complementary to a different subsequence of the first genomic target sequence of interest, the non-nucleotide unit positioned at a corresponding identical location of each of the probes of the first subset, and the label attached to the non-nucleotide unit; and

a second subset of oligonucleotide probes comprising at least 100 oligonucleotide probes for detecting a second genomic target of interest, each probe within the second subset having a non-nucleotide unit, a label, and an identical length, and each probe within the second subset further comprising a different nucleotide sequence complementary to a different subsequence of the second genomic target sequence of interest, the non-nucleotide unit positioned at a corresponding identical location of each of the probes of the second subset, and the label attached to the non-nucleotide unit.

13. The set of claim 12, wherein each probe of the first subset comprises the same label and each probe of the second subset comprises the same label, the label of each probe of the first subset being different than the label of each probe of the second subset.

14. The set of claim 12, wherein each probe of the first subset further comprises a plurality of non-nucleotide units, the plurality of non-nucleotide units being positioned at corresponding identical locations of each of the probes of the first subset.

15. 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, the incubating step being performed under conditions conducive to the binding of the set of oligonucleotide probes to the genomic target of interest; and

detecting hybridization of a plurality of the oligonucleotide probes of the set with the target sequence of interest in the sample, wherein the set of oligonucleotide probes comprises at least 100 oligonucleotide probes, each of the oligonucleotide probes having an identical length and having a different nucleotide sequence and being complementary to a different subsequence of the genomic target of interest, each of the oligonucleotide probes further comprising a non-nucleotide unit and at least one label, wherein the non-nucleotide unit is positioned at a corresponding identical location of each of the oligonucleotide probes and the at least one label is attached to the non-nucleotide unit.

16. The method according to claim 15, wherein the detecting step comprises one of a chromogenic reaction and a metallographic reaction when the label is suitable for detection by the chromogenic reaction or the metallographic reaction.

17. The method according to claim 16, wherein the incubating step is completed in less than 91 minutes.

18. The method according to claim 15, wherein the incubating and detecting steps comprise an in situ hybridization assay.

19. The method according to claim 15 further comprising the step of:

generating the set of oligonucleotide probes by designing the set of oligonucleotide probes and synthesizing the set of oligonucleotide probes such that the set comprises the least 100 oligonucleotide probes complementary to at least 100 different subsequences of the genomic target sequence of interest, and the non-nucleotide unit is incorporated to the oligonucleotide probes of the set.

20. The method according to claim 19, wherein each of the oligonucleotide probes further comprises a phosphoramidite modified building block, and the generating step includes attaching the at least one label to the non-nucleotide unit of each of the oligonucleotide probes and attaching the phosphoramidite modified building block to each of the oligonucleotide probes.

21. The method according to claim 20, wherein said synthesizing the set of oligonucleotide probes comprises phosphoramidite based oligonucleotide synthesis and the at least one label is attached to the non-nucleotide unit via an oxidation step of the phosphoramidite based oligonucleotide synthesis.

22. 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 a non-nucleotide unit, at least one label, and an identical length, and each of the oligonucleotide probes further comprising a different nucleotide sequence complementary to a different subsequence of the genomic target sequence of interest, the non-nucleotide unit positioned at a corresponding identical location of each of the oligonucleotide probes, and the at least one label attached to the non-nucleotide unit, and a detection agent.

23. The kit of claim 22, wherein the 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 an in situ hybridization assay.