Methods and compositions for detection and quantitation of nucleic acid analytes

• US 20040009506A1
• Filed: 03/28/2003
• Published: 01/15/2004
• Est. Priority Date: 03/29/2002
• Status: Abandoned Application

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Tan Wong, Wai Lee, Tsai, Siao Ping, Billeci, Todd, Stephan, Jean-Philippe F.
• Application Number:
  US10/401,520
• Publication Number:
  US 20040009506A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2537/125 Sandwich assay format
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide

Abstract

The present invention provides novel solution phase hybridization-based methods for detecting and quantitating nucleic acid analytes. Methods comprising use of novel capture polymers and/or signaling systems are provided. Use of these novel capture polymers and/or signaling systems provides significant improvements in signal to noise ratio, specificity, sensitivity and ease of development and use as compared to existing solution phase nucleic acid detection and quantitation methods. The invention further provides compositions, kits and articles of manufacture for practicing methods of the present invention.

Claims

1. A method for detecting or quantitating a nucleic acid analyte in a sample, said method comprising:

(A) contacting said sample with an analyte-binding oligonucleotide, a labeled oligonucleotide, a capture polymer and a linear stem oligonucleotide under conditions whereby a complex is formed comprising the analyte, analyte-binding oligonucleotide, labeled oligonucleotide, capture polymer and linear stem oligonucleotide, wherein;

(i) the analyte-binding oligonucleotide comprises (a) a sequence that is hybridizable to the analyte and (b) a sequence that is hybridizable to the stem oligonucleotide;

(ii) the linear stem oligonucleotide comprises (a) a sequence that is hybridizable to the analyte-binding oligonucleotide and (b) a sequence that is directly or indirectly hybridizable to the labeled oligonucleotide;

(iii) the labeled oligonucleotide comprises (a) a sequence that is directly or indirectly hybridizable to the stem oligonucleotide and (b) a label capable of directly or indirectly generating a detectable signal;

(iv) the capture polymer comprises a nucleic acid sequence that is directly or indirectly hybridizable to the analyte;

(B) detecting or quantitating the complex of step (A);

whereby detection or quantitation of the complex of step (A) is indicative of presence or quantity of the nucleic acid analyte in the sample.

16. The method of any of claims 1-15, further comprising contacting the sample with a blocker oligonucleotide.

17. The method of any of claims 1-15, wherein the capture polymer is hybridized to an oligonucleotide that is directly attached to a solid or semi-solid support.

18. The method of any of claims 1-15, wherein the capture polymer is directly attached to a solid or semi-solid support.

19. The method of any of claims 1-15, wherein the nucleic acid analyte is selected from the group consisting of RNA, DNA, RNA/DNA hybrid and nucleic acid-protein complex.

20. The method of any of claims 1-15, wherein the nucleic acid analyte comprises a sequence encoding part or all of a polypeptide selected from the group consisting of growth hormone, insulin-like growth factors, human growth hormone, N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, glycoprotein hormones, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), leutinizing hormone (LH), hematopoietic growth factor, vesicular endothelial growth factor (VEGF), hepatic growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-alpha, tumor necrosis factor-beta, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor, integrin, nerve growth factors (NGFs), NGF-beta, platelet-growth factor, transforming growth factors (TGFs), TGF-alpha, TGF-beta, insulin-like growth factor-I, insulin-like growth factor-II, erythropoietin (EPO), osteoinductive factors, interferons, interferon-alpha, interferon-beta, interferon-gamma, colony stimulating factors (CSFs), macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), granulocyte-CSF (G-CSF), thrombopoietin (TPO), interleukins (ILs), IL-1, IL-1alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, LIF, SCF, neurturin (NTN), kit-ligand (KL), HER2, human Fc, human heavy and light chains (constant region), KDR, nitric oxide synthase (NOS) and angiotensin converting enzyme (ACE).

21. The method of any of claims 1-15, wherein the sample is selected from the group consisting of blood, serum, sputum, urine, semen, cerebrospinal fluid, bronchial aspirate, organ tissue, cell lysate and cell culture medium.

22. The method of any of claims 1-15, wherein the sequence of the analyte-binding oligonucleotide that is hybridizable to the analyte is a sequence that is complementary to a sequence of the analyte.

28. The method of any of claims 1-15, wherein the label of the labeled oligonucleotide is selected from the group consisting of an antigen, a member of a specific binding pair, a fluorescent dye and a member of a reporter-quencher pair.

29. The method of claim 28, wherein said antigen is selected from the group consisting of digoxigenin, biotin and fluorescein isothiocyanate.

30. The method of claim 28, wherein said specific binding pair is selected from the group consisting of a receptor-ligand pair and an enzyme-substrate pair.

31. The method of claim 28, wherein said fluorescent dye is fluorescein isothiocyanate, rhodamine or Texas Red.

32. The method of claim 28, wherein the reporter-quencher pair comprises dyes capable of fluorescent resonance energy transfer.

33. The method of any of claims 1-15, wherein the labeled oligonucleotide is detected by contacting the labeled oligonucleotide with a compound that binds to the labels of the labeled oligonucleotide, wherein said compound is capable of directly or indirectly generating a detectable signal.

34. The method of any of claims 1-15, wherein capture polymers are provided as an array.

2. A method for detecting or quantitating a nucleic acid analyte in a sample, said method comprising:

(A) contacting said sample with an analyte-binding oligonucleotide, a linear labeled oligonucleotide and a capture polymer under conditions whereby a complex is formed comprising the analyte, analyte-binding oligonucleotide, linear labeled oligonucleotide and capture polymer, wherein;

(i) the analyte-binding oligonucleotide comprises (a) a sequence that is hybridizable to the analyte and (b) a sequence that is hybridizable to the linear labeled oligonucleotide;

(ii) the linear labeled oligonucleotide comprises (a) two or more units of label each attached directly to the oligonucleotide and (b) a sequence that is hybridizable to the analyte-binding oligonucleotide;

(iii) the capture polymer comprises a nucleic acid sequence that is directly or indirectly hybridizable to the analyte;

(B) detecting or quantitating the complex of step (A);

whereby detection or quantitation of the complex of step (A) is indicative of presence or quantity of the nucleic acid analyte in the sample.

3. A method for detecting or quantitating a nucleic acid analyte in a sample, said method comprising:

(A) contacting said sample with an analyte-binding linear labeled oligonucleotide and a capture polymer under conditions whereby a complex is formed comprising the analyte, analyte-binding linear labeled oligonucleotide and capture polymer, wherein;

(i) the analyte-binding linear labeled oligonucleotide comprises (a) a sequence that is hybridizable to the analyte and (b) two or more units of label each attached directly to the oligonucleotide;

(ii) the capture polymer comprises a nucleic acid sequence that is directly or indirectly hybridizable to the analyte;

(B) detecting or quantitating the complex of step (A);

whereby detection or quantitation of the complex of step (A) is indicative of presence or quantity of the nucleic acid analyte in the sample.

4. A method for detecting or quantitating a nucleic acid analyte in a sample, said method comprising:

(a) contacting the sample with an analyte-binding oligonucleotide and a capture polymer under conditions whereby a complex is formed comprising the analyte, analyte-binding oligonucleotide, and capture polymer, wherein;

(i) the analyte-binding oligonucleotide comprises a sequence that is hybridizable to the analyte; and

(ii) the capture polymer comprises a first portion that is hybridizable to the analyte and a second portion comprising a material that is not substantially hybridizable to nucleic acid;

(b) detecting or quantitating the complex of step (a);

whereby detection or quantitation of the complex of step (a) is indicative of presence or quantity of the nucleic acid analyte in the sample.

5. A method for detecting or quantitating a nucleic acid analyte in a sample, said method comprising:

(a) contacting the sample with an analyte-binding oligonucleotide and a capture polymer under conditions whereby a complex is formed comprising the analyte, analyte-binding oligonucleotide, and capture polymer, wherein;

(i) the analyte-binding oligonucleotide comprises a sequence that is hybridizable to the analyte; and

(ii) the capture polymer comprises a sequence that is hybridizable to the analyte and further comprises at least one modified nucleotide that enhances strength of hybridization of the polymer to the analyte;

(b) detecting or quantitating the complex of step (a);

whereby detection or quantitation of the complex of step (a) is indicative of presence or quantity of the nucleic acid analyte in the sample.

6. A method for detecting or quantitating a nucleic acid analyte in a sample, said method comprising:

(a) contacting the sample with an analyte-binding oligonucleotide and a capture polymer under conditions whereby a complex is formed comprising the analyte, analyte-binding oligonucleotide, and capture polymer, wherein;

(i) the analyte-binding oligonucleotide comprises a sequence that is hybridizable to the analyte; and

(ii) the capture polymer comprises a first portion that is hybridizable to the analyte, said first portion comprising at least one modified nucleotide that enhances strength of hybridization of the polymer to the analyte, and a second portion comprising a material that is not substantially hybridizable to nucleic acid;

(b) detecting or quantitating the complex of step (a);

whereby detection or quantitation of the complex of step (a) is indicative of presence or quantity of the nucleic acid analyte in the sample.

7. A method for detecting or quantitating a nucleic acid analyte in a sample, said method comprising:

(A) contacting said sample with an analyte-binding oligonucleotide, a labeled oligonucleotide, a capture polymer and a linear stem oligonucleotide under conditions whereby a complex is formed comprising the analyte, analyte-binding oligonucleotide, labeled oligonucleotide, capture polymer and linear stem oligonucleotide, wherein;

(i) the analyte-binding oligonucleotide comprises (a) a sequence that is hybridizable to the analyte and (b) a sequence that is hybridizable to the stem oligonucleotide;

(ii) the linear stem oligonucleotide comprises (a) a sequence that is hybridizable to the analyte-binding oligonucleotide and (b) a sequence that is directly or indirectly hybridizable to the labeled oligonucleotide;

(iii) the labeled oligonucleotide comprises (a) a sequence that is directly or indirectly hybridizable to the stem oligonucleotide and (b) a label capable of directly or indirectly generating a detectable signal;

(iv) the capture polymer comprises a first portion that is hybridizable to the analyte and and a second portion comprising a material that is not substantially hybridizable to nucleic acid;

(B) detecting or quantitating the complex of step (A);

whereby detection or quantitation of the complex of step (A) is indicative of presence or quantity of the nucleic acid analyte in the sample.

8. A method for detecting or quantitating a nucleic acid analyte in a sample, said method comprising:

(A) contacting said sample with an analyte-binding oligonucleotide, a linear labeled oligonucleotide and a capture polymer under conditions whereby a complex is formed comprising the analyte, analyte-binding oligonucleotide, linear labeled oligonucleotide and capture polymer, wherein;

(i) the analyte-binding oligonucleotide comprises (a) a sequence that is hybridizable to the analyte and (b) a sequence that is hybridizable to the linear labeled oligonucleotide;

(ii) the linear labeled oligonucleotide comprises (a) two or more units of label each attached directly to the oligonucleotide and (b) a sequence that is hybridizable to the analyte-binding oligonucleotide;

(iii) the capture polymer comprises a first portion that is hybridizable to the analyte and a second portion comprising a material that is not substantially hybridizable to nucleic acid;

(B) detecting or quantitating the complex of step (A);

whereby detection or quantitation of the complex of step (A) is indicative of presence or quantity of the nucleic acid analyte in the sample.

9. A method for detecting or quantitating a nucleic acid analyte in a sample, said method comprising:

(A) contacting said sample with an analyte-binding linear labeled oligonucleotide and a capture polymer under conditions whereby a complex is formed comprising the analyte, analyte-binding linear labeled oligonucleotide and capture polymer, wherein;

(i) the analyte-binding linear labeled oligonucleotide comprises (a) a sequence that is hybridizable to the analyte and (b) two or more units of label each attached directly to the oligonucleotide;

(ii) the capture polymer comprises a first portion that is hybridizable to the analyte and a second portion comprising a material that is not substantially hybridizable to nucleic acid;

(B) detecting or quantitating the complex of step (A);

whereby detection or quantitation of the complex of step (A) is indicative of presence or quantity of the nucleic acid analyte in the sample.

10. A method for detecting or quantitating a nucleic acid analyte in a sample, said method comprising:

(A) contacting said sample with an analyte-binding oligonucleotide, a labeled oligonucleotide, a capture polymer and a linear stem oligonucleotide under conditions whereby a complex is formed comprising the analyte, analyte-binding oligonucleotide, labeled oligonucleotide, capture polymer and linear stem oligonucleotide, wherein;

(i) the analyte-binding oligonucleotide comprises (a) a sequence that is hybridizable to the analyte and (b) a sequence that is hybridizable to the stem oligonucleotide;

(ii) the linear stem oligonucleotide comprises (a) a sequence that is hybridizable to the analyte-binding oligonucleotide and (b) a sequence that is directly or indirectly hybridizable to the labeled oligonucleotide;

(iii) the labeled oligonucleotide comprises (a) a sequence that is directly or indirectly hybridizable to the stem oligonucleotide and (b) a label capable of directly or indirectly generating a detectable signal;

(iv) the capture polymer comprises a nucleic acid sequence that is hybridizable to the analyte and further comprises at least one modified nucleotide that enhances strength of hybridization of the polymer to the analyte;

(B) detecting or quantitating the complex of step (A);

whereby detection or quantitation of the complex of step (A) is indicative of presence or quantity of the nucleic acid analyte in the sample.

45. The method of any of claims 5, 6 and 10-15, wherein the capture polymer comprises at least 3 said modified nucleotide.

46. The method of claim 45, wherein the capture polymer comprises at least 5 said modified nucleotide.

47. The method of claim any of claims 5, 6 and 10-15, wherein at least 10 percent of the total number of nucleotides in the capture polymer are said modified nucleotide.

48. The method of claim 47, wherein at least 20 percent of the total number of nucleotides in the capture polymer are said modified nucleotide.

49. The method of claim 48, wherein at least 30 percent of the total number of nucleotides in the capture polymer are said modified nucleotide.

50. The method of claim 49, wherein at least 40 percent of the total number of nucleotides in the capture polymer are said modified nucleotide.

51. The method of claim 50, wherein at least 50 percent of the total number of nucleotides in the capture polymer are said modified nucleotide.

52. The method of claim 47, wherein from about 10 to about 50 percent of the total number of nucleotides in the capture polymer are said modified nucleotide.

53. The method of any of claims 5, 6 and 10-15, wherein the modified nucleotide is 2′

-O-methoxy-RNA or derivative thereof.

54. The method of any of claims 5, 6 and 10-15, wherein at least one modified nucleotide is located in each of the 5′

and 3′

regions of the sequence that is hybridizable to the analyte.

11. A method for detecting or quantitating a nucleic acid analyte in a sample, said method comprising:

(A) contacting said sample with an analyte-binding oligonucleotide, a linear labeled oligonucleotide and a capture polymer under conditions whereby a complex is formed comprising the analyte, analyte-binding oligonucleotide, linear labeled oligonucleotide and capture polymer, wherein;

(i) the analyte-binding oligonucleotide comprises (a) a sequence that is hybridizable to the analyte and (b) a sequence that is hybridizable to the linear labeled oligonucleotide;

(ii) the linear labeled oligonucleotide comprises (a) two or more units of label each attached directly to the oligonucleotide and (b) a sequence that is hybridizable to the analyte-binding oligonucleotide;

(iii) the capture polymer comprises a nucleic acid sequence that is hybridizable to the analyte and further comprises at least one modified nucleotide that enhances strength of hybridization of the polymer to the analyte;

(B) detecting or quantitating the complex of step (A);

whereby detection or quantitation of the complex of step (A) is indicative of presence or quantity of the nucleic acid analyte in the sample.

23. The method of any of claims 2, 3, 5, 6, 8, 9, 11, 12, 14 and 15, wherein two tandem units of label of the linear labeled oligonucleotide are separated by at least about 1, 3 or 5 nucleotides.

24. The method of any of claims 2, 3, 5, 6, 8, 9, 11, 12, 14 and 15, wherein two tandem units of label of the linear labeled oligonucleotide are separated by from about 1 to about 12 nucleotides.

25. The method of any of claims 2, 3, 5, 6, 8, 9, 11, 12, 14 and 15, wherein two tandem units of label of the linear labeled oligonucleotide are separated by from about 3 to about 10 nucleotides.

26. The method of any of claims 2, 3, 5, 6, 8, 9, 11, 12, 14 and 15, wherein two tandem units of label of the linear labeled oligonucleotide are separated by from about 5 to about 8 nucleotides.

27. The method of any of claims 2, 3, 5, 6, 8, 9, 11, 12, 14 and 15, wherein the label is attached by covalent bond to the linear labeled oligonucleotide.

12. A method for detecting or quantitating a nucleic acid analyte in a sample, said method comprising:

(A) contacting said sample with an analyte-binding linear labeled oligonucleotide and a capture polymer under conditions whereby a complex is formed comprising the analyte, analyte-binding linear labeled oligonucleotide and capture polymer, wherein;

(i) the analyte-binding linear labeled oligonucleotide comprises (a) a sequence that is hybridizable to the analyte and (b) two or more units of label each attached directly to the oligonucleotide;

(ii) the capture polymer comprises a nucleic acid sequence that is hybridizable to the analyte and further comprises at least one modified nucleotide that enhances strength of hybridization of the polymer to the analyte;

(B) detecting or quantitating the complex of step (A);

whereby detection or quantitation of the complex of step (A) is indicative of presence or quantity of the nucleic acid analyte in the sample.

13. A method for detecting or quantitating a nucleic acid analyte in a sample, said method comprising:

(A) contacting said sample with an analyte-binding oligonucleotide, a labeled oligonucleotide, a capture polymer and a linear stem oligonucleotide under conditions whereby a complex is formed comprising the analyte, analyte-binding oligonucleotide, labeled oligonucleotide, capture polymer and linear stem oligonucleotide, wherein;

(i) the analyte-binding oligonucleotide comprises (a) a sequence that is hybridizable to the analyte and (b) a sequence that is hybridizable to the stem oligonucleotide;

(ii) the linear stem oligonucleotide comprises (a) a sequence that is hybridizable to the analyte-binding oligonucleotide and (b) a sequence that is directly or indirectly hybridizable to the labeled oligonucleotide;

(iii) the labeled oligonucleotide comprises (a) a sequence that is directly or indirectly hybridizable to the stem oligonucleotide and (b) a label capable of directly or indirectly generating a detectable signal;

(iv) the capture polymer comprises a first portion that is hybridizable to the analyte, said first portion comprising at least one modified nucleotide that enhances strength of hybridization of the polymer to the analyte, and a second portion comprising a material that is not substantially hybridizable to nucleic acid;

(B) detecting or quantitating the complex of step (A);

whereby detection or quantitation of the complex of step (A) is indicative of presence or quantity of the nucleic acid analyte in the sample.

35. The method of any of claims 4, 6-9 and 13-15, wherein the material that is not substantially hybridizable to nucleic acid is inert carbon.

36. The method of claim 35, wherein the inert carbon is provided as ethylene glycol.

37. The method of claim 36, wherein said ethylene glycol has the chemical structure 18-O-Dimethoxytritylhexaethyleneglycol, 1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.

38. The method of any of claims 4, 6-9 and 13-15, wherein the capture polymer comprises a spacer component.

39. The method of claim 38, wherein the spacer component comprises at least one C18 spacer.

40. The method of claim 39, wherein the spacer component comprises at least three C18 spacers.

41. The method of claim 40, wherein the spacer component comprises at least four C18 spacers.

42. The method of claim 39, wherein the spacer component comprises from about 1 to about 8 C18 spacers.

43. The method of claim 42, wherein the spacer component comprises from about 3 to about 6 C18 spacers.

44. The method of claim 38, wherein the spacer component is the material that is not substantially hybridizable to nucleic acid of the second portion of the capture polymer.

14. A method for detecting or quantitating a nucleic acid analyte in a sample, said method comprising:

(A) contacting said sample with an analyte-binding oligonucleotide, a linear labeled oligonucleotide and a capture polymer under conditions whereby a complex is formed comprising the analyte, analyte-binding oligonucleotide, linear labeled oligonucleotide and capture polymer, wherein;

(i) the analyte-binding oligonucleotide comprises (a) a sequence that is hybridizable to the analyte and (b) a sequence that is hybridizable to the linear labeled oligonucleotide;

(ii) the linear labeled oligonucleotide comprises (a) two or more units of label each attached directly to the oligonucleotide and (b) a sequence that is hybridizable to the analyte-binding oligonucleotide;

(iii) the capture polymer comprises a first portion that is hybridizable to the analyte, said first portion comprising at least one modified nucleotide that enhances strength of hybridization of the polymer to the analyte, and a second portion comprising a material that is not substantially hybridizable to nucleic acid;

(B) detecting or quantitating the complex of step (A);

whereby detection or quantitation of the complex of step (A) is indicative of presence or quantity of the nucleic acid analyte in the sample.

15. A method for detecting or quantitating a nucleic acid analyte in a sample, said method comprising:

(A) contacting said sample with an analyte-binding linear labeled oligonucleotide and a capture polymer under conditions whereby a complex is formed comprising the analyte, analyte-binding linear labeled oligonucleotide and capture polymer, wherein;

(i) the analyte-binding linear labeled oligonucleotide comprises (a) a sequence that is hybridizable to the analyte and (b) two or more units of label each attached directly to the oligonucleotide;

(ii) the capture polymer comprises a first portion that is hybridizable to the analyte, said first portion comprising at least one modified nucleotide that enhances strength of hybridization of the polymer to the analyte, and a second portion comprising a material that is not substantially hybridizable to nucleic acid;

(B) detecting or quantitating the complex of step (A);

whereby detection or quantitation of the complex of step (A) is indicative of presence or quantity of the nucleic acid analyte in the sample.