Electrochemical detection of mismatch nucleic acids

• US 20040018492A1
• Filed: 06/21/2002
• Published: 01/29/2004
• Est. Priority Date: 06/21/2001
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Huang, Jun, Wang, Tza-Huei, Miller, Jeffrey F., Ho, Chih-Ming, Liu, Minghsun
• Granted Patent: US 7,291,457 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2537/125 Sandwich assay format
• CPC Class Codes: C12Q 2563/113 the label being electroactive, e.g. redox labels
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode

Abstract

A method and system for specifically detecting perfectly matched probe-target hybrids by annealing a hairpin-based probe to a nucleic acid target under conditions favoring the formation of perfectly matched targets over mismatched targets, and electrochemically detecting the annealed probe. Sensitivity of detection can be enhanced by catalyzed reporter deposition of probe signal prior to electrochemical detection. Specificity can be enhanced by including hairpin-based competitor probes in the annealing step. The detection assay is particularly applicable to typing single nucleotide polymorphisms by DNA sandwich hybridization.

Claims

1. A method of specifically detecting perfectly matched probe-target hybrids, comprising the steps of:

annealing a hairpin-based probe to a nucleic acid target under conditions favoring the formation of perfectly matched probe-target hybrids over mismatched probe-target hybrids; and

electrochemically detecting annealed probe signal.

2. The method of claim 1 in which the probe is labeled with an electrochemically detectable molecule.

3. The method of claim 2 in which the probe is labeled with fluorescein.

4. The method of claim 1 in which the probe is labeled at one end with an electrochemically detectable molecule and attached at the other end to a molecule inhibiting electrochemical detection of the electrochemically detectable molecule.

5. The method of claim 4 in which the probe is labeled at one end with horse radish peroxidase and attached at the other end to benzhydroxamic acid.

6. The method of claim 1 in which the annealing step comprises incubating the probe and target in the presence of at least one hairpin-based competitor probe.

7. The method of claim 1 further comprising an amplification step after annealing and before detection, the step comprising amplifying annealed probe signal by catalyzed reporter deposition.

13. A method of typing a single nucleotide polymorphism of a target sequence, comprising applying the method of claim 1 to at least two hairpin-based probes, one probe forming a perfectly matched hybrid with the target sequence, each probe annealed in a separate reaction and comprising a sequence complementary to the target sequence, the complementary sequences of the probes differing from one another at a single nucleotide position.

14. The method of claim 13 further comprising an amplification step after annealing and before detection, the step comprising amplifying annealed probe signal by catalyzed reporter deposition.

8. A method of specifically detecting perfectly matched probe-target hybrids, comprising the steps of:

annealing a hairpin-based detector probe to a nucleic acid target under conditions favoring the formation of perfectly matched probe-target hybrids over mismatched probe-target hybrids; and

electrochemically detecting annealed probe signal by horse radish peroxidase-catalyzed reduction of an electrochemically active substrate.

9. The method of claim 8 in which the annealing step comprises incubating the detector probe and target in the presence of at least one hairpin-based competitor probe.

10. The method of claim 8 in which the detector probe is labeled with fluorescein.

11. The method of claim 8 in which the detector probe is labeled at one end with horse radish peroxidase and attached at the other end to benzhydroxamic acid.

12. The method of claim 8 further comprising an amplification step after annealing and before detection, the step comprising amplifying annealed probe signal by catalyzed reporter deposition of a tyramide conjugate.

15. A method of typing a single nucleotide polymorphism of a nucleic acid target sequence, comprising the steps of:

annealing each of at least two hairpin-based detector probes in separate reactions to the target sequence under conditions favoring the formation of perfectly matched probe-target hybrids over mismatched probe-target hybrids, one probe forming a perfectly matched hybrid with the target sequence, each probe comprising a sequence complementary to the target sequence, the complementary sequences of the probes differing from one another at a single nucleotide position; and

electrochemically detecting annealed probe signal by horse radish peroxidase-catalyzed reduction of an electrochemically active substrate.

16. The method of claim 15 in which the annealing step comprises incubating each probe and target in the presence of at least one hairpin-based competitor probe.

17. The method of claim 15 in which each detector probe is labeled with fluorescein.

18. The method of claim 15 in which each detector probe is labeled at one end with horse radish peroxidase and attached at the other end to benzhydroxamic acid.

19. The method of claim 15 further comprising an amplification step after annealing and before detection, the step comprising amplifying annealed probe signal by catalyzed reporter deposition of a tyramide conjugate.

20. A method of detecting an electrochemically detectable molecule, comprising:

amplifying molecular signal by catalyzed reporter deposition; and

electrochemically detecting the amplified signal.

21. The method of claim 20 in which the electrochemically detectable molecule is a nucleic acid probe annealed to a nucleic acid target.

22. The method of claim 20 in which the molecular signal is amplified by catalyzed reporter deposition of a tyramide conjugate.

23. A method of detecting a nucleic acid probe annealed to a nucleic acid target, comprising the steps of:

amplifying annealed probe signal by catalyzed reporter deposition of a tyramide-conjugate; and

electrochemically detecting the amplified probe signal by horse radish peroxidase-catalyzed reduction of an electrochemically active substrate.

24. A system for specifically detecting perfectly matched probe-target hybrids, comprising:

a hairpin-based probe for annealing to a nucleic acid target under conditions favoring the formation of perfectly matched probe-target hybrids over mismatched probe-target hybrids; and

a sensor for electrochemically detecting annealed probe signal.

25. The system of claim 24 in which the probe is labeled with an electrochemically detectable molecule.

26. The system of claim 25 in which the probe is labeled with fluorescein.

27. The system of claim 24 in which the probe is labeled at one end with an electrochemically detectable molecule and attached at the other end to a molecule inhibiting electrochemical detection of the electrochemically detectable molecule.

28. The system of claim 27 in which the probe is labeled at one end with horse radish peroxidase and attached at the other end to benzhydroxamic acid.

29. The system of claim 24 further comprising at least one hairpin-based competitor probe for incubating with the target.

30. The system of claim 24 further comprising an enzymatic system for amplifying annealed probe signal by catalyzed reporter deposition prior to electrochemical detection.

36. A system for typing a single nucleotide polymorphism of a nucleic acid target sequence, comprising adding at least one additional hairpin-based probe to the system of claim 24, each probe for annealing in a separate reaction and comprising a sequence complementary to the target sequence, the complementary sequences of the probes differing from one another at a single nucleotide position, one probe forming a perfectly matched hybrid with the target sequence.

37. The system of claim 36 further comprising at least one hairpin-based competitor probe for incubating with the target.

38. The system of claim 36 in which each probe is labeled with an electrochemically detectable molecule.

39. The system of claim 36 in which each probe is labeled at one end with an electrochemically detectable molecule and attached at the other end to a molecule inhibiting electrochemical detection of the electrochemically detectable molecule

40. The system of claim 36 further comprising an enzymatic system for amplifying annealed probe signal by catalyzed reporter deposition prior to electrochemical detection.

31. A system for specifically detecting perfectly matched probe-target hybrids, comprising:

a hairpin-based detector probe for annealing to a nucleic acid target under conditions favoring the formation of perfectly matched probe-target hybrids over mismatched probe-target hybrids; and

a sensor for electrochemically detecting annealed probe signal by horse radish peroxidase-catalyzed reduction of an electrochemically active substrate.

32. The system of claim 31 further comprising at least one hairpin-based competitor probe for incubating with the detector probe and the target.

33. The system of claim 31 in which the detector probe is labeled with fluorescein.

34. The system of claim 31 in which the detector probe is labeled at one end with horse radish peroxidase and attached at the other end to benzhydroxamic acid.

35. The system of claim 31 further comprising an enzymatic system for amplifying annealed probe signal by catalyzed reporter deposition of a tyramide conjugate prior to electrochemical detection.

41. A system for typing a single nucleotide polymorphism of a nucleic acid target sequence, comprising:

at least two hairpin-based detector probes for annealing in separate reactions to the target sequence under conditions favoring the formation of perfectly matched probe-target hybrids over mismatched probe-target hybrids, each probe comprising a sequence complementary to the target sequence, the complementary sequences of the probes differing from one another at a single nucleotide position, one probe forming a perfectly matched hybrid with the target sequence; and

a sensor for electrochemically detecting annealed probe by horse radish peroxidase-catalyzed reduction of an electrochemically active substrate.

42. The system of claim 41 further comprising at least one hairpin-based competitor probe for incubating with each detector probe and target.

43. The system of claim 41 in which the detector probe is labeled with fluorescein.

44. The system of claim 41 in which the detector probe is labeled at one end with horse radish peroxidase and attached at the other end to benzhydroxamic acid.

45. The system of claim 41 further comprising an enzymatic system for amplifying annealed probe signal by catalyzed reporter deposition of a tyramide conjugate prior to electrochemical detection.

46. A system for detecting an electrochemically detectable molecule, comprising:

an enzymatic system for amplifying molecular signal by catalyzed reporter deposition; and

a sensor for electrochemically detecting amplified signal.

47. The system of claim 46 in which the molecule is a nucleic acid probe annealed to a nucleic acid target.

48. The system of claim 46 in which the enzymatic system amplifies molecular signal by catalyzed reporter deposition of a tyramide conjugate.

49. A system for detecting a nucleic acid probe annealed to a nucleic acid target, comprising:

an enzymatic system for amplifying annealed probe signal by catalyzed reporter deposition of a tyramide conjugate; and

a sensor for electrochemically detecting annealed probe signal by horse radish peroxidase-catalyzed reduction of an electrochemically active substrate.