Reverse transcription on microarrays

• US 20040067492A1
• Filed: 10/04/2002
• Published: 04/08/2004
• Est. Priority Date: 10/04/2002
• Status: Abandoned Application

• Current Assignee: Mergen Ltd.
• Original Assignee: Mergen Ltd.
• Inventors: Love, James Robert Jr., Hu, Qianjin, Peng, Allan
• Application Number:
  US10/264,378
• Publication Number:
  US 20040067492A1
• 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 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: G01N 2035/00158 Elements containing microarrays, i.e. "biochip"

Abstract

The present invention provides novel methods for detection of gene. A method for detecting RNA molecules of interest in which oligonucleotide primers uniquely complementary to specific RNA molecules are used as primers for reverse transcribing target RNAs with reverse transcriptase, or any enzyme that possesses reverse transcription activity, is provided. The invention eliminates the need for labeling or converting the target RNA by incorporating detectable labels in the elongation product of reverse transcription of the probe-bound target RNA in the sample.

Claims

1. A method for detecting a target RNA in a sample, the method comprising,

(a) providing an array comprising a DNA primer comprising a sequence complementary to the target RNA;

(b) contacting the primer with an RNA sample under conditions that promote specific hybridization between the primer and the target RNA;

(c) incubating the primer-RNA heteroduplex with a reverse transcriptase enzyme, under conditions that allow reverse transcription of the RNA into cDNA such that a detectable label is incorporated into the cDNA; and

(d) detecting the detectable label incorporated into the nascent cDNA strand synthesized by the reverse transcriptase, wherein the presence of the label in the cDNA is indicative of the presence of the target RNA in the sample.

2. The method of claim 1, further comprising:

(e) comparing the presence or absence of a target RNA in a sample cell with respect to that in a reference cell.

3. The method of claim 1, further comprising:

(e) determining a level of target RNA in a sample cell and comparing with the level in a reference cell.

4. The method of claim 1, wherein a plurality of DNA primers is provided for detecting a plurality of target RNAs in a sample, wherein each primer is complementary to a different target RNA molecule and further wherein each primer is located at a distinct and identifiable location on a solid support.

5. The method of claim 1, wherein the detectable label is selected from the group consisting of a radiolabeled molecule, a fluorescent molecule, and a chromogenic molecule.

6. The method of claim 5, wherein the detectable label is incorporated into a modified nucleotide, further wherein the modified nucleotide is not a chain-terminating nucleotide.

7. The method of claim 6, wherein the detectable label is at least one of radiolabeled-dNTP, fluoro-dNTP, biotinylated dNTP or a digoxigenin-dNTP.

8. The method of claim 1, wherein the detectable label is conjugated to a molecule that binds a second label incorporated into the nascent polynucleotide.

9. The method of claim 1, wherein the array comprises from at least about 100 to at least about 1,000,000 primers.

10. The method of claim 9, wherein the array comprises at least about 1,000 primers.

11. The method of claim 1, wherein the sample is derived from a cell and the presence of at least one target RNA is indicative of a condition selected from the group consisting of an infection, a disease state, a predisposition to a disease state, a developmental, a physical, a chemical and a biological state.

12. The method of claim 1, wherein the sample is derived from a cell and the absence of at least one target RNA is indicative of a condition selected from the group consisting of an infection, a disease state, a predisposition to a disease state, a developmental, a physical, a chemical and a biological state.

13. The method of claim 1, wherein the primer is immobilized on a substrate.

14. The method of claim 13, wherein the primer is immobilized on the substrate by a covalent bond, such that the 3′

end of the primer is free.

15. The method of claim 14, wherein the covalent bond is selected from the group consisting of a Schiff base, a photocleavable bond, an electrostatic bond, a disulfide bond, a peptide bond, a diester bond, and a selectively releasable bond.

16. The method of claim 13, wherein the primer is immobilized on the substrate by a non-covalent coupling, such that the 3′

end of the primer is free.

17. The method of claim 16, wherein the non-covalent coupling is selected from the group consisting of an electrostatic interaction, a hydrogen bonds, an antibody-antigen coupling, a biotin-avidin interaction, a biotin-streptavidin interaction, a Staphylococcus aureus protein A-IgG antibody F_c fragment interaction, and a streptavidin/protein A chimera.

18. The method of claim 13, wherein the substrate is selected from the group consisting of a plastic, a ceramic, a nylon, a polyester, a metal, a resin, a gel, a membrane, a nitrocellulose membrane, a plate, a bead, a thin film, a glass, a cylinder, a tagged bead, a magnetic bead, an optical fiber, and a woven fiber.

19. The method of claim 1, wherein the RNA sample is isolated from a biological source.

20. The method of claim 19, wherein the RNA sample is amplified following isolation from a biological source.

21. The method of claim 1, wherein the RNA sample is prepared by transcription in vitro.

22. The method of claim 1, wherein the RNA sample comprises at least one expression control RNA.

23. The method of claim 1, wherein the primer further comprises a sequence for a promoter of a DNA-dependent RNA polymerase.

24. The method of claim 23, wherein the DNA-dependent RNA polymerase is selected from the group consisting of T3 RNA polymerase, T7 RNA polymerase, and SP6 RNA polymerase.

25. The method of claim 1, further comprising:

(e) amplifying the cDNA strand by a polymerase chain reaction (PCR) performed in situ.

26. The method of claim 25, wherein the PCR reaction is performed using a Taq DNA polymerase, a Tth1 DNA polymerase, a Vent DNA polymerase, a Pfu DNA polymerase, or a thermostable reverse transcriptase.

42. The method of claim 13, wherein the substrate is selected from the group consisting of a plastic, a ceramic, a nylon, a polyester, a metal, a resin, a gel, a membrane, a nitrocellulose membrane, a plate, a bead, a thin film, a glass, a cylinder, a tagged bead, a magnetic bead, an optical fiber, and a woven fiber.

27. A method for detecting a target RNA in a sample, the method comprising,

(a) providing a microarray comprising at least one DNA primer comprising a sequence complementary to a sequence of the target RNA;

(b) contacting the microarray with an RNA sample under conditions that allow specific hybridization between the primer and the target RNA;

(c) incubating the primer-RNA heteroduplex with a reverse transcriptase enzyme, under conditions that allow reverse transcription of the RNA into cDNA; and

(d) detecting the cDNA-RNA heteroduplex synthesized by the reverse transcriptase enzyme, wherein presence of the cDNA-RNA heteroduplex is indicative of the presence of the target RNA in the sample, wherein the detection is not mediated by an antibody against a DNA-RNA hybrid.

28. The method of claim 27, wherein the detectable label is conjugated to a molecule that binds the nascent polynucleotide.

29. The method of claim 27, further comprising:

(e) providing a thermostable reverse transcriptase; and

(f) performing a plurality of cycles of primer extension reactions under conditions wherein the temperature exceeds the melting temperature of a cDNA-RNA hybrid between each cycle.

30. The method of claim 27, wherein the cDNA-RNA heteroduplex is detected by a detectable reagent that specifically binds a double stranded polynucleotide.

31. The method of claim 30, wherein the detectable reagent is selected from the group consisting of an intercalating compound, a polynucleotide duplex-dependent fluorescence quenching compound, DAPI, ethidium bromide, thiazole orange, bis-benzimide and acridine orange.

32. The method of claim 30, wherein the detectable reagent is indirectly detectable by a second detectable molecule which binds the reagent.

33. The method of claim 27, wherein a plurality of DNA primers is provided for detecting a plurality of target RNAs in a sample, wherein each primer is complementary to a different target RNA molecule and further wherein each primer is located at a distinct and identifiable location on a solid support.

34. The method of claim 27, wherein the array comprises from at least about 100 to at least about 1,000,000 primers.

35. The method of claim 34, wherein the array comprises at least about 1,000 primers.

36. The method of claim 27, wherein the sample is derived from a cell and the presence of at least one target RNA is indicative of a condition selected from the group consisting of an infection, a disease state, a predisposition to a disease state, a developmental, a physical, a chemical and a biological state.

37. The method of claim 27, wherein the sample is derived from a cell and the absence of at least one target RNA is indicative of a condition selected from the group consisting of an infection, a disease state, a predisposition to a disease state, a developmental, a physical, a chemical and a biological state.

38. The method of claim 27, wherein the primer is immobilized on the substrate by a covalent bond, such that the 3′

end of the primer is free.

39. The method of claim 38, wherein the covalent bond is selected from the group consisting of a photocleavable bond, an electrostatic bond, a disulfide bond, a peptide bond, a diester bond, and a selectively releasable bond.

40. The method of claim 27, wherein the primer is immobilized on the substrate by a non-covalent coupling, such that the 3′

end of the primer is free.

41. The method of claim 40, wherein the non-covalent coupling is selected from the group consisting of an electrostatic interaction, a hydrogen bonds, an antibody-antigen coupling, a biotin-avidin interaction, a biotin-streptavidin interaction, a Staphylococcus aureus protein A-IgG antibody F_c fragment interaction, and a streptavidin/protein A chimera.

43. The method of claim 27, wherein the RNA sample is isolated from a biological source.

44. The method of claim 43, wherein the RNA sample is amplified following isolation from a biological source.

45. The method of claim 27, wherein the RNA sample is prepared by transcription in vitro.

46. The method of claim 27, wherein the RNA sample comprises at least one expression control RNA.

47. A method for detecting a target RNA in a sample, the method comprising,

(a) providing an array comprising a DNA primer comprising a sequence complementary to the target RNA;

(b) contacting the primer with an RNA sample under conditions that promote specific hybridization between the primer and the target RNA;

(c) incubating the primer-RNA heteroduplex with a thermostable reverse transcriptase enzyme, under conditions that allow reverse transcription of the RNA into cDNA such that a detectable label is incorporated into the cDNA;

(d) performing at least two cycles of hybridization and reverse transcription, under conditions comprising a temperature higher than a melting temperature of the RNA;

cDNA duplex; and

(e) detecting the detectable label incorporated into the nascent cDNA strand synthesized by the reverse transcriptase, wherein the presence of the label in the cDNA is indicative of the presence of the target RNA in the sample.

48. The method of claim 47, further comprising:

(f) comparing the presence or absence of a target RNA in a sample cell with respect to that in a reference cell.

49. The method of claim 47, wherein the hybridization and reverse transcription steps are performed in the presence of an RNase H inhibitor.

50. The method of claim 47, wherein the thermostable reverse transcriptase lacks an RNase H function.

51. A kit for identifying sequence variations in a target polynucleotide as compared to a reference sequence, comprising:

a) an array of at least two oligonucleotide primers immobilized on a solid phase support, wherein each oligonucleotide primer is selected to comprise a sequence complementary to a target RNA, and occupies an identifiable and discrete area of the array;

b) reagents suitable for a reverse transcription reaction on the array; and

c) detection means for detecting a nascent cDNA strand on the array.

52. The kit of claim 51, wherein the detection means comprises a detectable label that is incorporated into the nascent cDNA strand during the reverse transcription reaction.

53. The kit of claim 52, wherein the detectable label is a fluorescent molecule.

54. The kit of claim 52, wherein the detectable label comprises at least one of radiolabeled-dNTP, fluoro-dNTP, biotinylated dNTP or a digoxigenin-dNTP.