Set of oligonucleotide probes as well as methods and uses thereto

• US 10,640,815 B2
• Filed: 04/22/2016
• Issued: 05/05/2020
• Est. Priority Date: 07/30/2009
• Status: Active Grant

• Current Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Original Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Inventors: Bergmann, Frank, Eberle, Walter, Fischer, Thomas, von der Eltz, Herbert
• Primary Examiner(s): Lu, Frank W
• Application Number:
  US15/136,532
• Publication Number:
  US 20160230221A1
• Time in Patent Office: Days
• Field of Search: 435 61, 435 611, 435 911, 436 94, 436501, 536 231, 536 243, 536 2433
• 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 first set of oligonucleotide probes with a sample comprising a genomic target sequence of interest under conditions for the binding of the first set of oligonucleotide probes to the genomic target sequence of interest; and

detecting hybridizations of a plurality of the oligonucleotide probes of the first set of oligonucleotide probes with the target sequence of interest, thereby detecting the genomic target sequence of interest in the sample by one of a chromogenic reaction or a metallographic reaction,

wherein the first set of oligonucleotide probes comprises at least 100 different oligonucleotide probes, each of the oligonucleotide probes of the first set of oligonucleotide probes comprising a different nucleotide sequence complementary to a different subsequence of the genomic target sequence of interest, each of the oligonucleotide probes of the first set of oligonucleotide probes comprising at least three first labels, each of the at least three first labels attached to one of a base, a sugar, or a phosphate moiety of a nucleotide in each of the oligonucleotide probes of the first set of oligonucleotide probes at about equally spaced positions, and

wherein the detecting step comprises one of a chromogenic reaction or a metallographic reaction, and wherein the at least three first labels are the same for each of the oligonucleotide probes of the first set of oligonucleotide probes.

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 first 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 first 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 first 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 first labels in each of the oligonucleotide probes are positioned at locations separated by at least 16 nucleotides.

9. The method of claim 1, further comprising incubating a second set of oligonucleotide probes with the sample;

wherein the second set of oligonucleotide probes comprises at least 100 different oligonucleotide probes, each of the oligonucleotide probes of the second set of oligonucleotide probes having at least three second labels, wherein the at least three second labels coupled to each of the oligonucleotide probes of the second set of oligonucleotide probes are the same; and

wherein the at least three second labels coupled to each of the oligonucleotide probes of the second set of oligonucleotide probes are different than the at least three first labels coupled to each of the oligonucleotide probes of the first set of oligonucleotide probes.

10. 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 first set of oligonucleotide probes with a sample comprising the genomic target sequence of interest under conditions for the binding of the first set of oligonucleotide probes to the genomic target sequence of interest; and

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

wherein the first set of oligonucleotide probes comprises at least 100 different oligonucleotide probes, each of the oligonucleotide probes of the first set of oligonucleotide probes comprising a different nucleotide sequence complementary to a different subsequence of the genomic target sequence of interest, each of the oligonucleotide probes of the first set of oligonucleotide probes comprising at least three first labels, each of the at least three first labels attached to one of a base, a sugar, or a phosphate moiety of a nucleotide in each of the oligonucleotide probes of the first set of oligonucleotide probes at about equally spaced positions, and wherein the at least three first labels are the same for each of the oligonucleotide probes of the first set of oligonucleotide probes.

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

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

13. The method according to claim 10, wherein the detecting step comprises one of a chromogenic reaction or a metallographic reaction.

14. The method of claim 10, wherein the at least three first labels in each of the oligonucleotide probes are positioned at locations separated by at least 16 nucleotides.

15. The method of claim 10, wherein the at least three first labels include one of a hapten, a radioisotope, biotin, digoxigenin or a 2,4-dinitrophenyl moiety.

16. The method of claim 10, further comprising incubating a second set of oligonucleotide probes with the sample;

wherein the second set of oligonucleotide probes comprises at least 100 different oligonucleotide probes, each of the oligonucleotide probes of the second set of oligonucleotide probes having at least second three labels, wherein the at least three second labels coupled to each of the oligonucleotide probes of the second set of oligonucleotide probes are the same; and

wherein the at least three second labels coupled to each of the oligonucleotide probes of the second set of oligonucleotide probes are different than the at least three first labels coupled to each of the oligonucleotide probes of the first set of oligonucleotide probes.