Nucleic acid amplification and detection method

• US 20050287591A1
• Filed: 08/24/2005
• Published: 12/29/2005
• Est. Priority Date: 07/31/2002
• Status: Abandoned Application

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Browne, Kenneth A.
• Application Number:
  US11/211,231
• Publication Number:
  US 20050287591A1
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00313 the reactor vessels being formed by arrays of wells in blocks
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00677 Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/14 Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
• CPC Class Codes: C40B 60/14 for creating libraries

Abstract

Method of amplifying and detecting a nucleic acid target molecule contained in a test sample using a device having amplification primers and detectably labeled hybridization probes immobilized on the same surface of a solid support. Soluble amplicons produced in an in vitro nucleic acid amplification reaction employing the immobilized amplification primers are detected by the immobilized hybridization probes. Surface-immobilized amplification primers and hybridization probes are maintained in fluid communication throughout the procedure.

Claims

1. A method of determining whether a test sample contains a target nucleic acid comprising a target sequence, said method comprising the steps of:

providing a device for amplifying nucleic acids and detecting amplification products, said device comprising,

a solid support having a surface,

a plurality of species of amplification primer immobilized to said surface, said plurality of species of amplification primer comprising a first amplification primer that comprises a sequence complementary to a first strand of said target nucleic acid, and

a plurality of species of detectably labeled hybridization probe immobilized to said surface, said plurality of species of detectably labeled hybridization probe comprising a first hybridization probe that comprises a detectable label and a sequence of bases complementary to said target sequence;

contacting said plurality of species of amplification primer and said plurality of species of detectably labeled hybridization probe with said test sample;

synthesizing in an in vitro amplification reaction multiple copies of said target sequence and the complement thereof by a process comprising enzymatic extension of said first amplification primer immobilized to said surface, whereby there are produced soluble amplification products if said test sample contained said target nucleic acid; and

detecting hybridization of said soluble amplification products to said first hybridization probe immobilized to said surface, thereby determining that said test sample contained said target nucleic acid,

wherein each of said plurality of species of detectably labeled hybridization probe and said plurality of species of amplification primer is in fluid communication with the others during the synthesizing and detecting steps.

2. The method of claim 1, wherein no portion of said surface of said solid support is excluded from occupation by an immobilized oligonucleotide, said device having been manufactured by a process comprising immersion of said surface of said solid support in a liquid composition comprising immobilizable oligonucleotide primers.

3. The method of claim 2, wherein said solid support is a bead.

4. The method of claim 2, wherein said surface of said solid support is a planar surface.

5. The method of claim 4, wherein said first hybridization probe is one component in an array.

6. The method of claim 2, wherein the in vitro amplification reaction in the synthesizing step is an isothermal in vitro amplification reaction that does not require thermal cycling.

7. The method of claim 6, wherein said process comprising enzymatic extension in the synthesizing step comprises enzymatic extension by a reverse transcriptase.

8. The method of claim 7, wherein said soluble amplification products consist of RNA.

9. The method of claim 7, wherein said reverse transcriptase comprises an endogenous RNase H activity.

10. The method of claim 2, wherein said plurality of species of detectably labeled hybridization probe comprises no more than two species of detectably labeled hybridization probe.

11. The method of claim 10, wherein said plurality of species of detectably labeled hybridization probe that comprises no more than two species of detectably labeled hybridization probe comprises no more than a single species of detectably labeled hybridization probe.

12. The method of claim 2, wherein said plurality of species of amplification primer comprises no more than a single species of amplification primer having a free 3′

terminus available for extension by a DNA polymerase activity.

13. The method of claim 12, wherein said plurality of species of detectably labeled hybridization probe comprises no more than two species of detectably labeled hybridization probe.

14. The method of claim 11, wherein said plurality of species of amplification primer comprises no more than a single species of amplification primer having a free 3′

terminus available for extension by a DNA polymerase activity.

15. The method of claim 2, wherein each of said plurality of species of detectably labeled hybridization probe in said device comprises a fluorophore moiety and a quencher moiety.

16. The method of claim 15, wherein each of said plurality of species of detectably labeled hybridization probe that comprises a fluorophore moiety and a quencher moiety is a molecular beacon.

17. The method of claim 15, wherein each of said plurality of species of detectably labeled hybridization probe that comprises a fluorophore moiety and a quencher moiety is a molecular torch.

18. The method of claim 1, wherein said plurality of species of detectably labeled hybridization probe comprises no more than two species of detectably labeled hybridization probe.

19. The method of claim 18, wherein said plurality of species of detectably labeled hybridization probe that comprises no more than two species of detectably labeled hybridization probe comprises no more than a single species of detectably labeled hybridization probe.

20. The method of claim 19, wherein said plurality of species of amplification primer comprises no more than a single species of amplification primer having a free 3′

terminus available for extension by a DNA polymerase activity.

21. The method of claim 18, wherein said plurality of species of amplification primer comprises no more than a single species of amplification primer having a free 3′

terminus available for extension by a DNA polymerase activity.

22. The method of claim 18, wherein said solid support is a bead.

23. The method of claim 18, wherein said surface of said solid support is a planar surface.

24. The method of claim 1, wherein said process comprising enzymatic extension in the synthesizing step comprises enzymatic extension by a reverse transcriptase.

25. The method of claim 1, wherein the in vitro amplification reaction in the synthesizing step is an isothermal in vitro amplification reaction that does not require thermal cycling.

26. The method of claim 25, wherein said process comprising enzymatic extension in the synthesizing step comprises enzymatic extension by a reverse transcriptase.

27. The method of claim 26, wherein said soluble amplification products produced in the synthesizing step consist of RNA.

28. The method of claim 26, wherein said reverse transcriptase comprises an endogenous RNase H activity.

29. The method of claim 1, wherein each of said plurality of species of detectably labeled hybridization probe in said device comprises a fluorophore moiety and a quencher moiety.

30. The method of claim 29, wherein each of said plurality of species of detectably labeled hybridization probe that comprises a fluorophore moiety and a quencher moiety is a molecular beacon.

31. The method of claim 29, wherein each of said plurality of species of detectably labeled hybridization probe that comprises a fluorophore moiety and a quencher moiety is a molecular torch.

32. The method of claim 1, wherein the detecting step comprises detecting a fluorescent signal.

33. The method of claim 1, wherein said soluble amplification products produced in the synthesizing step consist of RNA.

34. The method of claim 1, wherein said solid support is a bead.

35. The method of claim 1, wherein said surface of said solid support is a planar surface.

36. The method of claim 35, wherein said first hybridization probe is one component in an array.