Device and method of detecting mutations and polymorphisms in DNA

• US 20050164236A1
• Filed: 09/22/2004
• Published: 07/28/2005
• Est. Priority Date: 09/22/2003
• Status: Active Grant

• Current Assignee: Agency For Science Technology and Research, National University of Singapore
• Original Assignee: Agency For Science Technology and Research, National University of Singapore
• Inventors: Robelek, Rudolf, Su, Xiaodi, Knoll, Wolfgang, O'Shea, Sean
• Granted Patent: US 8,105,780 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings

Abstract

There is provided a resonator sensor useful for detecting polymorphisms and mutations in DNA. The resonator sensor has a capture molecule immobilised on its surface, the capture molecule being either a probe DNA containing a reference sequence, or a mismatch binding molecule, and being capable of forming a probe DNA/target DNA/mismatch binding molecule complex on the surface of the resonator. A method for detecting mutations in a target DNA, including single nucleotide polymorphisms, is also provided.

Claims

1. A method of detecting a mutation in a target DNA comprising:

obtaining a first measurement of a resonance parameter of a resonator sensor, said resonator sensor having a vibrating element, said vibrating element having a surface, and a probe DNA immobilised on said surface, said probe DNA hybridized with a target DNA;

contacting the hybridized probe DNA and target DNA with a mismatch binding molecule;

obtaining a second measurement of the resonance parameter of the resonator sensor;

and comparing the first and second measurement of the resonance parameter.

2. The method of claim 1 wherein the resonator sensor comprises a bulk acoustic wave device, a quartz crystal microbalance, a surface acoustic wave device, a flexural wave plate, a piezoelectric thin film, a quartz tuning fork, a MEMS resonator, a membrane, a bridge, a suspended masses or a cantilever.

3. The method of claim 1 wherein obtaining comprises measuring resonant frequency, impulse response, resonance amplitude, phase, quality factor, damping property, energy dissipation, response spectrum or sharpness of resonance.

4. The method of claim 1 wherein the mutation is a single base substitution, a deletion of 1 to 4 bases, or an insertion of 1 to 4 bases.

5. The method of claim 1 wherein the target DNA is extracted from a cell from a patient.

6. The method of claim 5 wherein the cell is a blood cell, a skin cell, a liver cell, a kidney cell, a lung cell, a breast cell or a tumour cell.

7. The method of claim 1 wherein the mismatch binding molecule is a mismatch binding protein.

8. The method of claim 7 wherein the mismatch binding protein is MutS from Escherichia coli, Salmonella typhimurium or Thermus aquaticus, or HexA from Streptococcus pneumoniae.

9. The method of claim 1 wherein the mismatch binding molecule is a synthetic mismatch binding ligand.

10. The method of claim 9 wherein the synthetic mismatch binding ligand is a naphthyridine dimer, a naphthyridine-azaquinolone hybrid or an aminonaphthyridine dimer.

11. The method of claim 1 wherein the probe DNA comprises a non-disease sequence of a genetic marker.

12. The method of claim 11 wherein the genetic marker is a marker for cancer, atherosclerosis, heart disease, diabetes, cystic fibrosis or Alzheimer'"'"'s disease.

13. The method of claim 1 wherein the probe DNA is immobilized on the surface of the vibrating element through a reactive functional group attached at one end of the probe DNA.

14. The method of claim 13 wherein the reactive functional group is a thiol group, amino group, carboxylic acid group, hydroxyl group, phenol group or phosphate group.

15. The method of claim 1 wherein the probe DNA is immobilized on the surface of the vibrating element through an affinity binding molecule attached at one end of the probe DNA.

16. The method of claim 15 wherein the affinity binding molecule is biotin, streptavidin, avidin, an ATP analogue, an ATP binding domain, or a 6-histidine peptide.

17. The method of claim 1 wherein the mismatch binding molecule is modified with a heavy metal nanoparticle.

18. The method of claim 1 where the mismatch binding molecule is modified with an enzyme that cleaves a substrate to produce an insoluble product.

19. The method of claim 1 wherein the resonator sensor comprises a plurality of resonator sensors arranged in an array and a different probe DNA is immobilized on a surface of a vibrating element of each resonator sensor in the array, and wherein each probe DNA is hybridized with the same target DNA.

20. The method of claim 1 wherein the resonator sensor comprises a plurality of resonator sensors arranged in an array and the same probe DNA is immobilized on a surface of a vibrating element of each resonator sensor in the array, and wherein a different target DNA is hybridized with the probe DNA on said surface of said vibrating element of said each resonator sensor.

21. A method of detecting a mutation in a target DNA comprising:

obtaining a first measurement of a resonance parameter of a resonator sensor, said resonator sensor having a vibrating element, said vibrating element having a surface, and a mismatch binding molecule immobilised on said surface;

contacting the mismatch binding molecule with a hybridized probe DNA and target DNA;

obtaining a second measurement of the resonance parameter of the resonator sensor;

and comparing the first and second measurement of the resonance parameter.

22. The method of claim 21 wherein the resonator sensor comprises a bulk acoustic wave device, a quartz crystal microbalance, a surface acoustic wave device, a flexural wave plate, a piezoelectric thin film, a quartz tuning fork, a MEMS resonator, a membrane, a bridge, a suspended masses or a cantilever.

23. The method of claim 21 wherein obtaining comprises measuring resonant frequency, impulse response, resonance amplitude, phase, quality factor, damping property, energy dissipation, response spectrum or sharpness of resonance.

24. The method of claim 21 wherein the mutation is a single base substitution, a deletion of 1 to 4 bases, or an insertion of 1 to 4 bases.

25. The method of claim 21 wherein the target DNA is extracted from a cell from a patient.

26. The method of claim 25 wherein the cell is a blood cell, a skin cell, a liver cell, a kidney cell, a lung cell, a breast cell or a tumour cell.

27. The method of claim 21 wherein the mismatch binding molecule is a mismatch binding protein.

28. The method of claim 27 wherein the mismatch binding protein is MutS from Escherichia coli, Salmonella typhimurium or Thermus aquaticus, or HexA from Streptococcus pneumoniae.

29. The method of claim 21 wherein the mismatch binding molecule is a synthetic mismatch binding ligand.

30. The method of claim 29 wherein the synthetic mismatch binding ligand is a naphthyridine dimer, a naphthyridine-azaquinolone hybrid or an aminonaphthyridine dimer.

31. The method of claim 21 wherein the probe DNA comprises a non-disease sequence of a genetic marker.

32. The method of claim 31 wherein the genetic marker is a marker for cancer, atherosclerosis, heart disease, diabetes, cystic fibrosis or Alzheimer'"'"'s disease.

33. The method of claim 21 wherein the mismatch binding molecule is immobilized on the surface of the vibrating element of the resonator sensor through a reactive functional group on the mismatch binding molecule.

34. The method of claim 33 wherein the reactive functional group is a thiol group, amino group, carboxylic acid group, hydroxyl group, phenol group or phosphate.

35. The method of claim 21 wherein the mismatch binding molecule is immobilized on the surface of the vibrating element of the resonator sensor through an affinity binding molecule attached to the mismatch binding molecule.

36. The method of claim 35 wherein the affinity binding molecule is biotin, streptavidin, avidin, an ATP analogue, an ATP binding domain, or a 6-histidine peptide.

37. The method of claim 21 wherein the mismatch binding molecule is immobilized through physical adsorption to the surface of the vibrating element of the resonator sensor.

38. The method of claim 37 wherein the surface of the vibrating element is coated with a polymer film.

39. The method of claim 38 wherein the polymer film is nitrocellulose, polystyrene, polyethylene, or nylon.

40. The method of claim 21 wherein the probe DNA is modified with a heavy metal nanoparticle.

41. The method of claim 21 where the probe DNA is modified with an enzyme that cleaves a substrate to produce an insoluble product.

42. The method of claim 21 wherein the resonator sensor comprises a plurality of resonator sensors arranged in an array and a different mismatch binding molecule is immobilized on a surface of a vibrating element of each resonator sensor in the array and wherein the same hybridized probe DNA and target DNA is contacted with each mismatch binding molecule.

43. A resonator sensor comprising:

a vibrating element having a surface;

a probe DNA immobilized on said surface, said probe DNA capable of hybridizing with a target DNA;

an excitation source for vibrating said vibrating element; and

a detector for detecting a change in vibration of said vibrating element indicative of binding of a mismatch binding molecule to said probe DNA when hybridized with said target DNA to form a heteroduplex DNA.

44. The resonator sensor of claim 43 wherein said detecting comprises detecting a change in resonant frequency, impulse response, resonance amplitude, phase, quality factor, damping property, energy dissipation, response spectrum or sharpness of resonance.

45. The resonator sensor of claim 43 further comprising an additional layer on said surface.

46. The resonator sensor of claim 45 wherein said additional layer is metal or polymer film.

47. The resonator sensor of claim 46 wherein the polymer film is nitrocellulose, polystyrene, polyethylene, or nylon.

48. The resonator sensor of claim 43 wherein the probe DNA is immobilized on said surface through a reactive functional group attached at one end of the probe DNA.

49. The resonator sensor of claim 48 wherein said reactive functional group is a thiol group, amino group, carboxylic acid group, hydroxyl group, phenol group or phosphate group.

50. The resonator sensor of claim 43 wherein the probe DNA is immobilized on said surface through an affinity binding molecule attached at one end of said probe DNA.

51. The resonator sensor of claim 50 wherein the affinity binding molecule is biotin, streptavidin, avidin, an ATP analogue, an ATP binding domain, or a 6-histidine peptide.

52. The resonator sensor of claim 43 wherein the probe DNA comprises a non-disease sequence of a genetic marker.

53. The resonator sensor of claim 52 wherein the genetic marker is a marker for cancer, atherosclerosis, heart disease, diabetes, cystic fibrosis or Alzheimer'"'"'s disease.

54. A resonator sensor comprising:

a vibrating element having a surface;

a mismatch binding molecule on said surface, said mismatch binding molecule capable of binding a heteroduplex DNA;

an excitation source for vibrating said vibrating element;

a detector for detecting a change in vibration of said vibrating element indicative of binding of said heteroduplex DNA to said mismatch binding molecule.

55. The resonator sensor of claim 54 wherein said detecting comprises detecting a change in resonant frequency, impulse response, resonance amplitude, phase, quality factor, damping property, energy dissipation, response spectrum or sharpness of resonance.

56. The resonator sensor of claim 54 further comprising an additional layer on said surface.

57. The resonator sensor of claim 56 wherein said additional layer is metal or polymer film.

58. The resonator sensor of claim 57 wherein the polymer film is nitrocellulose, polystyrene, polyethylene, or nylon.

59. The resonator sensor of claim 54 wherein the mismatch binding molecule is a mismatch binding protein.

60. The resonator sensor of claim 54 wherein the mismatch binding protein is MutS from Escherichia coli, Salmonella typhimurium or Thermus aquaticus, or HexA from Streptococcus pneumoniae.

61. The resonator sensor of claim 60 wherein the mismatch binding protein is immobilized on said surface through physical adsorption.

62. The resonator sensor of claim 54 wherein the mismatch binding molecule is a synthetic mismatch binding ligand.

63. The resonator sensor of claim 62 wherein the synthetic mismatch binding ligand is naphthyridine, a naphthyridine-azaquinolone hybrid or an aminonaphthyridine dimer.

64. The resonator sensor of claim 54 wherein the mismatch binding molecule is immobilized on said surface through a reactive functional group on the mismatch binding molecule.

65. The resonator sensor of claim 64 wherein the reactive functional group is a thiol group, amino group, carboxylic acid group, hydroxyl group, phenol group or phosphate group.

66. The resonator sensor of claim 54 wherein the mismatch binding molecule is immobilized on said surface through an affinity binding molecule attached to the mismatch binding molecule.

67. The resonator sensor of claim 66 wherein the affinity binding molecule is biotin, streptavidin, avidin, an ATP analogue, an ATP binding domain, or a 6-histidine peptide.