Genomic assay

• US 8,486,622 B2
• Filed: 09/23/2005
• Issued: 07/16/2013
• Est. Priority Date: 09/24/2004
• Status: Expired due to Fees

• Current Assignee: Ingeneus Inc.
• Original Assignee: Ingeneus Inc.
• Inventors: Erikson, Glen H., Daksis, Jasmine
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US11/575,821
• Publication Number:
  US 20090123914A1
• Time in Patent Office: 2,853 Days
• Field of Search: 435/6, 435/6.1, 536/23.1, 536/24.3
• US Class Current: 435/6.1
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

A method of detecting a nucleic acid sequence in a genomic sample, includes: providing the genomic sample containing a target nucleic acid sequence of a duplex nucleic acid; providing, a probe containing a probe nucleic acid sequence; providing a hybridization mixture containing the genomic sample, the probe, a hybridization promoting agent and labels; incubating the hybridization mixture; irradiating the incubated mixture with radiation effective to stimulate at least some of the labels to emit energy; and detecting from a fluorescent signal whether the probe perfectly matches the target nucleic acid sequence, wherein the detecting is completed within sixty minutes of the hybridization mixture providing, and the method is conducted without denaturing and without PCR amplifying the duplex nucleic acid. A kit for practicing the method includes the probe, the hybridization promoting agent, and labels.

Claims

1. A method of detecting a nucleic acid sequence in a genomic sample, said method comprising:

providing the genomic sample comprising a quantity of duplex nucleic acids containing target nucleic acid sequences;

providing a quantity of probes comprising probe nucleic acid sequences;

providing a hybridization mixture comprising the genomic sample, the quantity of probes, a quantity of hybridization promoting agents and labels;

incubating the hybridization mixture to provide an incubated mixture comprising complexes of the duplex nucleic acids, the probes and the labels;

irradiating the incubated mixture with radiation effective to stimulate at least some of the labels to emit energy; and

detecting from a fluorescent signal whether the probe nucleic acid sequences perfectly match the target nucleic acid sequences, to thereby detect whether the nucleic acid sequence is present in the genomic sample,

wherein a ratio of the quantity of probes to the quantity of duplex nucleic acids is at least 10⁹, and the method is conducted without denaturing the duplex nucleic acids and without PCR amplification of the duplex nucleic acids.

2. The method of claim 1, wherein a single or multiple nucleotide polymorphism is detected.

3. The method of claim 1, wherein the detecting is completed within sixty minutes of providing the hybridization mixture.

4. The method of claim 3, wherein a haplotype is detected.

5. The method of claim 1, wherein a morphological status of an organism or cell from which the genomic sample was obtained is detected, said morphological status comprising at least one of information regarding a stage of development and information regarding a disease state.

6. The method of claim 1, wherein the quantity of the duplex nucleic acids in the genomic sample is less than 700 copies.

7. The method of claim 6, wherein the quantity of the duplex nucleic acids in the genomic sample is about 150 to about 300 copies.

8. The method of claim 6, wherein the genomic sample consists essentially of contents of a single cell.

9. The method of claim 1, wherein the detecting is conducted in a biological cell.

10. The method of claim 1, wherein the genomic sample is more than 5 kb in length.

11. The method of claim 1, wherein the genomic sample is undigested throughout the method.

12. The method of claim 1, wherein the nucleic acid sequence belongs to a pathogen present or previously present in an organism or a cell from which the genomic sample is obtained.

13. The method of claim 1, wherein each of the probes, if unitary, is a single-stranded nucleic acid or nucleic acid analogue of 15 to 30 bases in length.

14. The method of claim 1, wherein each of the hybridization promoting agents is an intercalating label.

15. The method of claim 14, wherein the intercalating label comprises dimeric cyanine dyes.

16. The method of claim 15, wherein the intercalating label consists of YOYO-1.

17. The method of claim 1, wherein the labels are intercalating fluorophores which also constitute the hybridization promoting agent.

18. The method of claim 1, wherein each of the hybridization promoting agents is a kosmotrope.

19. The method of claim 1, wherein each of the hybridization promoting agents is a cation of a compound selected from the group consisting of (CH₃)₄NCl, (CH₃)₃N.HCl, NaCl, Na₂SO₄, Na₂HPO₄, and (NH₄)₂SO₄.

20. The method of claim 1, wherein the labels are non-intercalating fluorophores.

21. The method of claim 1, wherein an incubation period is from one to ten minutes, and the method is completely practiced in less than fifteen minutes.

22. The method of claim 21, wherein the hybridization mixture is maintained at a temperature of 20 to 40°

C. throughout the incubation period.

23. The method of claim 1, wherein at least one base of the probes binds to a base or a base pair of the target nucleic acid sequences by Watson-Crick complementary base interaction and/or by homologous base interaction, such that each of the complexes is a triplex.

24. The method of claim 1, wherein at least one base of the probes binds to a base or a base pair of the target nucleic acid sequences by Watson-Crick complementary base interaction and/or by homologous base interaction, such that each of the complexes is a quadruplex.

25. The method of claim 1, wherein the probes and the target nucleic acid sequences do not bond together solely as antiparallel strands obeying Watson-Crick base pairing rules.

26. The method of claim 1, wherein the radiation is a laser beam having a power density of about 84 W/cm²/sec.

27. The method of claim 1, wherein the fluorescent signal is compared with a reference fluorescent signal to determine whether the probe nucleic acid sequences perfectly match the target nucleic acid sequences.

28. The method of claim 1, wherein the detecting comprises monitoring a change in the fluorescent signal over time to determine whether the probe nucleic acid sequences perfectly match the target nucleic acid sequences, and wherein an increase in the fluorescent signal over time indicates a perfect match and a decrease in the fluorescent signal over time indicates a lack of a perfect match.

29. The method of claim 1, wherein the probes are provided in the hybridization mixture in a target saturating amount, said target saturating amount being a probe concentration in excess of a target nucleic acid sequences concentration, and the labels are provided in the hybridization mixture in a complex saturating amount, said complex saturating amount being a label concentration above which discrimination of the signal from background signals changes at a first rate less than a second rate at which the label concentration changes.

30. The method of claim 1, wherein each of the probes has a length effective to optimize energy transfer or migration.

31. The method of claim 30, wherein discrimination of the fluorescent signal from background signals is maximized by energy transfer or energy migration between intra-target intercalated labels and probe-target intercalated labels.

32. The method of claim 30, wherein each of the probes is 20-30 bases or base pairs in length.

33. The method of claim 1, wherein a length of the probes is selected so as to maximize an intensity of the fluorescent signal.

34. The method of claim 1, wherein the probes are provided in the hybridization mixture in a target saturating amount, the labels are provided in the hybridization mixture in a complex saturating amount, and each of the probes has a length effective to optimize energy transfer or migration.

35. The method of claim 1, wherein a preliminary mixture comprising a buffer and water is subjected to an applied electric charge effective to enhance a sensitivity of the method, and at least a portion of the preliminary mixture is subsequently incorporated into the hybridization mixture.

36. The method of claim 1, wherein the genomic sample is added to the hybridization mixture after the probes, the hybridization promoting agents and the labels.

37. The method of claim 1, wherein the hybridization promoting agents or the labels are added to the hybridization mixture last.

38. The method of claim 1, wherein the genomic sample is purified, semipurified, unpurified or diluted.

39. The method of claim 1, wherein levels of gene product expression in a genomic sample are detected or gene product expressions of at least two genomic samples are compared.

40. The method of claim 1, wherein the hybridization mixture further comprises a plurality of additional probes that bind to sequences of the genomic sample adjacent to the target nucleic acid sequences.

41. The method of claim 1, wherein each of the probes comprises an emission quencher and at least one of the labels.

42. The method of claim 1, wherein each of the probes is modified by at least one linked moiety.

43. The method of claim 1, wherein the labels comprise FET, FRET, energy migration or redox sets.

44. The method of claim 1, wherein the labels comprise quantum dots.

45. The method of claim 1, wherein nucleic acid sequence repeats, insertions or deletions are detected.

46. The method of claim 1, wherein the detecting is repeated under varied conditions of the hybridization mixture.

47. The method of claim 1, wherein a cancerous or disease state of an organism or cell from which the genomic sample was obtained is detected, or pregnancy of the organism is determined.

48. The method of claim 1, wherein the genomic sample is obtained from a human sample of tissue, buccal cells, blood, fluid, sputum, urine or feces, and labeled with a molecular identification tag adapted to identify a source of the genomic sample.

49. The method of claim 1, wherein the probes are provided on a support selected from the group consisting of a bead, a plate, a membrane, a film, a microwell, an electrode, a column and a capillary tube.

50. The method of claim 1, wherein the probes are provided on a silver island film.

51. A kit for practicing the method of claim 1, said kit comprising:

a support selected from the group consisting of a bead, a plate, a membrane, a film, a microwell, an electrode, a column and a capillary tube, said support being adapted to hold the quantity of genomic sample comprising the quantity of duplex nucleic acids;

,

the probes provided on the support;

the hybridization promoting agents; and

the labels,

wherein the ratio of the quantity of probes provided on the support to the quantity of duplex nucleic acids is at least 10⁹.

52. The kit of claim 51, further comprising a container in which the hybridization mixture is provided.

53. The kit of claim 52, wherein the container is adapted to collect sputum or the kit further comprises a sputum collection device.

54. The kit of claim 52, wherein the container comprises a molecular identification tag adapted to identify a source of the genomic sample.

55. The kit of claim 51, wherein the support contains 10¹² probes.

56. The kit of claim 51, wherein the support is a silver island film.

57. The kit of claim 51, wherein each of the probes is single-stranded and contains a hairpin, or is double-stranded.

58. The kit of claim 51, wherein the labels comprise dimeric cyanine dyes.

59. The kit of claim 51, wherein the labels and the hybridization promoting agents consist of YOYO-1.

60. The kit of claim 51, wherein each of the hybridization promoting agents is at least one compound selected from the group consisting of (CH₃)₄NCl, (CH₃)₃N.HCl, NaCl, Na₂SO₄, Na₂HPO₄, and (NH₄)₂SO₄.

63. The method of claim 1, wherein subsequent to the incubating step and prior to the applying energy step, the incubated mixture is further incubated under conditions such that the probes disassociate from the target nucleic acid sequences, and wherein the detecting step comprises determining from the signal whether the probes are disassociated from the target nucleic acid sequences and the conditions of incubation, so as to determine whether the probes perfectly match the target nucleic acid sequences.

64. The method of claim 1, wherein each of the probes comprises one or more parts, and at least one of the parts is 5 to 30 bases in length.

65. The method of claim 1, wherein a separation step is performed before the irradiating step.

66. The method of claim 1, further comprising a step of providing at least one blocking probe to suppress binding of the probes to a non-target sequence of the genomic sample.

67. The method of claim 1, wherein the complex acts as a photonic structure for collecting photonic energy and transferring energy to a signal emitting label.

68. The method of claim 1, wherein the steps of the method are repeated more than once to provide more than one hybridization mixture and more than one fluorescent signal, provided that each hybridization mixture is formed by combining the genomic sample, the probes, the hybridization promoting agents and the labels in a different sequence.

69. The method of claim 1, wherein the detecting comprises monitoring a change in fluorescence anisotropy of the fluorescent signal over time to determine whether the probes perfectly match the target nucleic acid sequences.

61. A method of detecting a nucleic acid sequence in a genomic sample, said method comprising:

providing the genomic sample comprising a quantity of single-stranded or double-stranded nucleic acids containing target nucleic acid sequences;

providing a quantity of probes comprising probe nucleic acid sequences;

providing a hybridization mixture comprising the genomic sample, the quantity of probes, a quantity of hybridization promoting agents and labels;

incubating the hybridization mixture to provide an incubated mixture comprising a complex of the single-stranded or double-stranded nucleic acids, the probes and the labels;

applying energy to the incubated mixture effective to elicit a signal from the hybridization mixture; and

detecting from the signal whether the probe nucleic acid sequences perfectly match the target nucleic acid sequences, to thereby detect whether the nucleic acid sequence is present in the genomic sample,

wherein;

(a) a ratio of the quantity of probes to the quantity of nucleic acids is at least 10⁹, (b) the detecting is completed within sixty minutes of providing of the hybridization mixture, and (c) the method is conducted without denaturing the duplex nucleic acid and without PCR amplification of the duplex nucleic acid.

62. The method of claim 61, wherein subsequent to the incubating step and prior to the applying energy step, the incubated mixture is further incubated under conditions such that the probes disassociate from the target nucleic acid sequences, and wherein the detecting step comprises determining from the signal whether the probes are disassociated from the target nucleic acid sequences and the conditions of incubation, so as to determine whether the probes perfectly match the target nucleic acid sequences.