HIGH THROUGHPUT DETECTION OF MICRORNAS AND USE FOR DISEASE DIAGNOSIS

• US 20090181390A1
• Filed: 10/31/2008
• Published: 07/16/2009
• Est. Priority Date: 01/11/2008
• Status: Abandoned Application

• Current Assignee: Signosis, Inc.
• Original Assignee: Signosis, Inc.
• Inventors: Jiang, Xin, Li, Xianqiang
• Application Number:
  US12/263,152
• Publication Number:
  US 20090181390A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/207 siRNA, miRNA
• CPC Class Codes: C12Q 2525/313 Branched oligonucleotides
• CPC Class Codes: C12Q 2561/125 Ligase Detection Reaction [LDR]

Abstract

Methods, compositions and kits are provided for high throughput detection of micro RNAs (miRNA), especially for sensitive and specific detection of miRNA that are in low abundance and closely related to each other. In particular, an assembly of designed oligonucleotide probes with unique tag sequences is used to achieve these purposes via high throughput microarrays, optionally in conjunction with branched-DNA based array detection. The assays can be used for diagnosis, prognosis or monitoring of diseases or disorders such as cancer, for pharmacogenomic studies of patient stratification and drug responses, for discovery of therapeutic targets, or for forensic analysis.

Claims

1. A method for detecting a target nucleic acid in a sample comprising

providing a sample potentially containing the target nucleic acid

providing at least one oligonucleotide probe set, wherein said probe set comprises:

(i) a first oligonucleotide probe having a 5′

target specific region and a first 3′

universal sequence region,

(ii) a second oligonucleotide probe having a 3′

target specific region and a second 5′

universal sequence region

(iii) a third oligonucleotide probe having a 5′

region complementary to said first 3′

universal sequence region in said first oligonucleotide probe, and

(v) a fourth oligonucleotide probe having a 3′

region complementary to the 5′

universal sequence region of said second oligonucleotide probe

wherein the first and said second oligonucleotides probes are suitable for ligation together when hybridized adjacent to one another to said target nucleic acid, and wherein the third and the fourth oligonucleotides probes are suitable for ligation to the target nucleic acid when hybridized adjacent to said nucleic acid target;

annealing the oligonucleotide probe set to the target nucleic acid such that a complex is formed between the target nucleic acid and the oligonucleotide probe set;

contacting the complex with a linking agent under conditions such that the directly adjacent 5′ and

3′

ends of the first and second oligonucleotide probes, the 3′ and

5′

ends of the third oligonucleotide probe and the target nucleic acid, and the 5′ and

3′

ends of the fourth oligonucleotide probe and the target nucleic acid covalently bond to form a ligated probe product,

separating the ligated probe product from the non-ligated first and second oligonucleotide probes; and

detecting whether or not said ligated probe product is formed, wherein the presence of the ligated probe product is indicative of presence of said target nucleic acid in said sample.

2. The method of claim 1 wherein the oligonucleotide probes in said oligonucleotide probe set have a predetermined sequence.

3. The method of claim 1 wherein said first oligonucleotide probe comprises in a 3′

to 5′

order said universal region, a tag region and said target specific region.

4. The method of claim 1 wherein said second oligonucleotide probe comprises in a 3′

to 5′

order said target specific region, a tag region and said universal sequence region.

5. The method of claim 3 wherein said third oligonucleotide probe comprises a 3′

region that is complementary to the tag region of said first oligonucleotide probe.

6. The method of claim 3 further comprising a fifth oligonucleotide probe that is complementary to the tag region of said first oligonucleotide probe.

7. The method of claim 4 wherein said fourth oligonucleotide probe comprises a 5′

region that is complementary to the tag region of said second oligonucleotide probe.

8. The method of claim 4 further comprising a fifth oligonucleotide probe that is complementary to the tag region of said second oligonucleotide probe.

9. The method of claim 1 wherein at least one of the universal regions of the first and the second oligonucleotide probe is a promoter sequence.

10. The method of claim 9 wherein the promoter sequence is used as a primer of DNA polymerase.

11. The method of claim 10 wherein said DNA polymerase is selected from the group consisting of Thermoanaerobacter thermohydrosulfuricus DNA polymerase, Thermococcus litoralis DNA polymerase I, E. coli DNA polymerase I, Taq DNA polymerase I, Tth DNA polymerase I, Bacillus stearothermophilus (Bst) DNA polymerase I, E. coli DNA polymerase III, bacteriophage T5 DNA polymerase, bacteriophage M2 DNA polymerase, bacteriophage T4 DNA polymerase, bacteriophage T7 DNA polymerase, bacteriophage phi29 DNA polymerase, bacteriophage PRD1 DNA polymerase, bacteriophage phi15 DNA polymerase, bacteriophage phi21DNA polymerase, bacteriophage PZE DNA polymerase, bacteriophage PZA DNA polymerase, bacteriophage Nf DNA polymerase, bacteriophage M2Y DNA polymerase, bacteriophage B103 DNA polymerase, bacteriophage SF5 DNA polymerase, bacteriophage GA-1 DNA polymerase, bacteriophage Cp-5 DNA polymerase, bacteriophage Cp-7 DNA polymerase, bacteriophage PR4DNA polymerase, bacteriophage PR5 DNA polymerase, bacteriophage PR722 DNA polymerase and bacteriophage L17 DNA polymerase.

12. The method of claim 9 wherein the promoter sequence is a promoter for a phage polymerase.

13. The method of claim 12 wherein said phage polymerase is selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase or SP6 RNA polymerase.

14. The method of claim 1 further comprising annealing a first primer complementary to the universal sequence region of the first oligonucleotide probe, contacting the annealed primer with a polymerase under conditions such that the annealed primer is extended to form an extension product complementary to the sequences to which the primers is annealed.

15. The method of claim 14 further comprising detecting the presence of said extension product, wherein the presence of the extended product is indicative of the presence of said target nucleic acid in said sample.

16. The method of claim 14 further comprising annealing a second primer complementary to the universal sequence region of the fourth oligonucleotide probe, contacting the annealed second primer with a polymerase under conditions such that the annealed primer is extended to form an extension product complementary to the sequences to which the primers is annealed.

17. The method of claim 16 further comprising detecting the presence of said extension product, wherein the presence of the extended product is indicative of the presence of the target nucleic acid in said sample.

18. The method of claim 1 further comprising annealing a first primer complementary to the universal sequence region of the fourth oligonucleotide probe, contacting the annealed primer with a polymerase under conditions such that the annealed primer is extended to form extension products complementary to the sequences to which the primers is annealed.

19. The method of claim 18 further comprising detecting the presence of said extension product, wherein the presence of the extended product is indicative of the presence of said target nucleic acid in said sample.

20. The method of claim 15, 16 or 19 wherein said extension product is detected using a DNA microarray, bead microarray, high throughput sequencing or single microtiter plate assay.

21. The method of claim 18 wherein said extension product has a detectable label.

22. The method of claim 21 wherein said detectable label is a fluorescent or biotin label, and the step of detecting includes detecting a fluorescent signal generated by the fluorescent, chemiluminescent or color.

23. The method of claim 21 wherein said label is attached to said primer complementary to said universal sequence region of said first oligonucleotide probe.

24. The method of claim 21 wherein said label is incorporated during the extension of said annealed primer complementary to the universal sequence region of said first oligonucleotide probe.

25. The method of claim 24 wherein said incorporation includes adding a label nucleotide to the extension of the annealed primer complementary to the universal sequence region of said third oligonucleotide probe.

26. The method of claim 1 wherein said universal sequence region of said second oligonucleotide is a phage promoter.

27. The method of claim 26 wherein said phage promoter is selected from the group consisting of T7 RNA polymerase promoter, T3 RNA polymerase promoter or SP6 RNA polymerase promoter.

28. The method of claim 26 further comprising

contacting the phage promoter region of the second oligonucleotide probe with a phage polymerase under conditions such that a transcription product of said phage promoter region is formed

detecting the presence of the transcription product, wherein the presence of the transcription product is indicative of the presence of the target nucleic acid in the sample.

30. The method of claim 28, wherein said transcription product is detected using a DNA microarray, bead microarray, high throughput sequencing or a single microtiter plate assay.

31. The method of claim 28, wherein the transcription product has a detectable label.

32. The method of claim 31 wherein said detectable label is a fluorescent or biotin label, and the step of detecting includes detecting a fluorescent signal generated by the fluorescent or chemiluminescent or color

39. The method of claim 31 wherein said label is incorporated during the transcription of said phage promoter region of said second oligonucleotide probe.

40. The method of claim 39 wherein said incorporation includes adding a label nucleotide to the transcription of said phage promoter region of said second oligonucleotide probe.

41. The method of claim 28 wherein said target nucleic acid is a miRNA molecule.

42. The method of claim 41 wherein said miRNA molecule is derived from total RNA

43. The method of claim 1 wherein said first or third oligonucleotide further comprises a capturing portion.

44. The method of claim 43 wherein said capturing portion is used to separate the ligated probe product from unligated first and second oligonucleotide probes.

45. The method of claim 43 wherein said capturing portion is biotin or a capture sequence.

46. The method of claim 45 wherein said capturing portion is biotin.

47. The method of claim 46 wherein said ligated probe product is isolated by binding said biotin with a strepavidin bound to a solid support.

48. The method of claim 3 or 4 wherein said tag region in said first oligonucleotide probe or said tag in said second oligonucleotide probe are specifically assigned to the target nucleic acid.

49. The method of claim 1 further comprising a loop that links said second and said fourth oligonucleotide.

50. The method of claim 49 further comprising detecting the presence of the ligated probe containing said loop to indicate the presence of said target nucleic acid in said sample.

52. The method of claim 50 wherein said ligated probe is detected using a DNA microarray, bead microarray, or high throughput sequencing.

51. The method claim 506 wherein said detecting comprises binding a branched DNA to said ligated probe.