Claims
- 1. A method for characterizing a nucleic acid molecule, comprising:
providing a nucleic acid molecule for characterization; modifying a property of at least one defined local area of the nucleic acid molecule, wherein the defined local area comprises at least two consecutive nucleotides; contacting the modified nucleic acid with a substrate, the substrate including a detector capable of identifying a characteristic of the nucleic acid molecule and the detector being responsive to the modification of the nucleic acid molecule; causing the modified nucleic acid molecule to traverse a defined volume on the substrate so that individual nucleotides of the modified nucleic acid molecule interact with the detector in sequential order, whereby data correlating with the modification of the nucleic acid molecule are obtained.
- 2. The method of claim 1, wherein two more or local areas of the nucleic acid molecule are modified.
- 3. The method of claim 1, wherein the defined local area comprises no more than fifty consecutive nucleotides.
- 4. The method of claim 1, wherein the defined local area comprises no more than thirty consecutive nucleotides.
- 5. The method of claim 1, wherein the defined local area comprises no more than twenty consecutive nucleotides.
- 6. The method of claim 1, wherein the defined local area comprises no more than ten consecutive nucleotides.
- 7. The method of claim 1, wherein the property of the local area to be modified is selected from the group consisting of local bulk, local chemistry, local charge, local charge density, and the local electronic current carrying capacity of the molecule.
- 8. The method of claim 7, wherein the local defined area is modified by increasing the total bulk of the local defined area.
- 9. The method of claim 7, wherein the local defined area is modified by altering the charge of the defined local area of the nucleic acid molecule.
- 10. The method of claim 7, wherein the local defined area is modified by altering the charge density of the defined local area of the nucleic acid molecule.
- 11. The method of claim 7, wherein the local defined area is modified by altering the local electronic current carrying capacity of the defined local area of the nucleic acid molecule
- 12. The method of claim 1 or 2, wherein the modifying step is accomplished by chemically modifying a nucleotide of defined local area of the nucleic acid molecule.
- 13. The method of claim 12, wherein the base moiety of the nucleotide is modified.
- 14. The method of claim 12, wherein the sugar moiety of the nucleotide is modified.
- 15. The method of claim 12, wherein the phosphate moiety of the nucleotide is modified.
- 16. The method of claim 12, wherein the chemical modifier comprises a covalently linked tag, said tag capable of generating an identifiable signal upon interaction with the detector.
- 17. The method of claim 1 or 2, wherein the modifying step is accomplished by non-covalently binding a probe to the nucleic acid molecule.
- 18. The method of claim 17, wherein the probe comprises an oligonucleotide.
- 19. The method of claim 18, wherein the oligonucleotide comprises at least one modified nucleotide.
- 20. The method of claim 17, wherein the probe comprises a plurality of oligonucleotides.
- 21. The method of claim 20, wherein the oligonucleotides comprise at least one modified nucleotide.
- 22. The method of claim 17, wherein the probe comprises a sequence specific protein.
- 23. The method of claim 22, wherein the sequence-specific protein is a Zn2+ finger protein.
- 24. The method of claim 17, wherein the probe comprises a plurality of sequence specific proteins having a plurality of binding specificities.
- 25. The method of claim 1, wherein the nucleic acid molecule is single stranded.
- 26. The method of claim 1, wherein the nucleic acid molecule is double stranded.
- 27. The method of claim 1, wherein the nucleic acid molecule comprises genomic DNA.
- 28. The method of claim 17, wherein the probe is chosen to bind a specific sequence of a nucleic acid molecule.
- 29. The method of claim 28 wherein the sequence contains a single nucleotide polymorphism (SNP).
- 30. A method for characterizing a nucleic acid molecule, said method comprising:
(a) providing;
(i) a sample comprising at least one nucleic acid molecule for characterization; and (ii) a probe capable of binding to a specific nucleic acid sequence; (b) combining the sample and the probe under conditions such that the probe binds to nucleic acid molecule to form a nucleic acid:probe complex at locations where the specific nucleic acid sequence is present; (c) detecting the presence and location of binding in the nucleic acid:probe complex by:
(i) contacting the nucleic acid:probe complex with a substrate, the substrate including a detector capable of identifying a characteristic of a nucleic acid molecule; and (ii) causing the nucleic acid:probe complex to traverse a defined volume of the substrate so that nucleotides of the nucleic acid interact with the detector in sequential order, whereby data correlating with the presence and/or location of binding are obtained.
- 31. The method of claim 30 wherein the probe comprises a sequence-specific binding protein.
- 32. The method of claim 31, wherein the probe comprises at least one Zn2+ finger protein.
- 33. The method of claim 31, wherein the probe comprises at least two Zn finger proteins, and wherein the each of the Zn2+ finger proteins comprises a moiety that promotes cooperative binding to DNA.
- 34. The method of claim 31, wherein the probe comprises a plurality of Zn2+ finger proteins.
- 35. The method of claim 31, wherein the probe comprises dimeric Zn2+ finger proteins.
- 36. The method of claim 30, wherein the probe comprises at least one oligonucleotide.
- 37. The method of claim 36, wherein the oligonucleotide is a genome-specific oligonucleotide.
- 38. The method of claim 36, wherein the oligonucleotide is an allele-specific oligonucleotide.
- 39. The method of claim 36, wherein the probe comprises a set of universal oligonucleotides.
- 40. The method of claim 36, wherein the oligonucleotide is an mRNA specific oligonucleotide.
- 41. The method of claim 40, wherein the oligonucleotide comprises a nucleotide sequence complementary to an mRNA splice junction.
- 42. The method of claim 30, wherein the probe binds to the nucleic acid molecule at multiple sites to form multiple nucleic acid:probe complexes.
- 43. The method of claim 42, wherein the probe comprises a sequence-specific binding protein.
- 44. The method of claim 43, wherein the probe comprises at least one Zn2+ finger protein.
- 45. The method of claim 43, wherein the probe comprises at least two Zn2+ finger proteins, and wherein the each of the Zn2+ finger proteins comprises a moiety that promotes cooperative binding to DNA.
- 46. The method of claim 43, wherein the probe comprises dimeric Zn2+ finger proteins.
- 47. The method of claim 42, wherein the probe comprises at least one oligonucleotide.
- 48. The method of claim 47, wherein the oligonucleotide is a genome-specific oligonucleotide.
- 49. The method of claim 47, wherein the oligonucleotide is an allele-specific oligonucleotide.
- 50. The method of claim 47, wherein the probe comprises a set of universal oligonucleotides.
- 51. The method of claim 47, wherein the oligonucleotide is a mRNA specific oligonucleotide.
- 52. The method of claim 51, wherein the oligonucleotide comprises a nucleotide sequence corresponding to an mRNA splice junction.
- 53. A method of characterizing a nucleic acid molecule, the method comprising:
(a) providing a sample containing at least one nucleic acid molecule for characterization; (b) modifying a property of a plurality of defined local areas of the nucleic acid molecule under conditions wherein the connectivity of the nucleic acid molecule is maintained, wherein the defined local area comprises one or more nucleotides; and (c) detecting the presence and locations of the defined local areas under conditions wherein the connectivity of the nucleic acid molecule is maintained, whereby data correlating with the presence and/or locations of defined local areas are obtained.
- 54. The method of claim 53, wherein the modifying step is accomplished by increasing the cross sectional volume of the defined local areas of the nucleic acid molecule.
- 55. The method of claim 53, wherein the modifying step is accomplished by chemically modifying at least one nucleotide in each of the defined local areas of the nucleic acid molecule.
- 56. The method of claim 55, wherein the chemical modifier comprises a covalently linked tag, said tag capable of generating an identifiable signal upon interaction with the detector.
- 57. The method of claim 53, wherein the modifying step is accomplished by non-covalently binding a probe to the nucleic acid molecule.
- 58. The method of claim 53, wherein the probe comprises an oligonucleotide.
- 59. The method of claim 58, wherein the oligonucletide comprises at least one modified nucleotide.
- 60. The method of claim 53, wherein the probe comprises a sequence-specific protein.
- 61. The method of 60, wherein the sequence-specific protein is a Zn2+ finger protein.
- 62. The method of claim 53, further providing a plurality of probes, each probe capable of binding to or modifying a specific nucleic acid sequence, and combining the sample and the plurality of probes under conditions wherein the connectivity of the nucleic acid molecule is maintained and wherein the probes bind to the nucleic acid to form nucleic acid: probe complexes at multiple sites on the nucleic acid molecule where the specific nucleic acid sequence is present; and
detecting the presence and locations of probe:nucleic acid complexes under conditions wherein the connectivity of the nucleic acid molecule is maintained.
- 63. The method of claim 62, wherein the probes comprise Zn2+ finger proteins.
- 64. The method of claim 62, wherein the probes comprise at least two Zn2+ finger proteins, and wherein the each of the Zn2+ finger proteins comprises a sequence that promotes binding to DNA.
- 65. The method of claim 62, wherein the probes comprise a plurality of Zn2+ finger proteins.
- 66. The method of claim 62, wherein the probes comprise dimeric Zn2+ finger proteins.
- 67. The method of claim 63, wherein the probes comprise oligonucleotides.
- 68. The method of claim 66, wherein the oligonucleotides are genome-specific oligonucleotides.
- 69. The method of claim 66, wherein the oligonucleotides are allele-specific oligonucleotides.
- 70. The method of claim 66, wherein the oligonucleotides are a set of universal oligonucleotides.
- 71. A method for characterizing a nucleic acid molecule, comprising:
generating a population of nucleic acid fragments from a target double stranded nucleic acid; characterizing the nucleic acid fragments by:
(a) contacting the nucleic acid fragment population with a surface, the surface including a detector capable of detecting the interaction time of nucleic acids; (b) causing the nucleic acid fragments to traverse a defined volume on the surface so that the nucleotides of a nucleic acid fragment interact with the detector in sequential order, whereby data correlated with a characteristic of the nucleic acid fragment are obtained.
- 72. The method of claim 71, wherein the step of characterizing the nucleic acid fragments includes determining the relative amount and/or length of the fragments.
- 73. The method of claim 71, wherein the population of nucleic acid fragments are characterized to provide a size distribution of nucleic acid fragments.
- 74. The method of claim 71, further comprising:
providing at least one probe capable of binding to a specific nucleic acid sequence; combining the nucleic acid fragments and the probe under conditions such that the probe binds to the nucleic acid fragments to form a nucleic acid: probe complex at locations where the specific nucleic acid sequence is present; wherein the detector is capable of identifying a characteristic of a nucleic acid fragment; and wherein data correlating with the presence and/or location of binding to the nucleic acid fragments are obtained.
- 75. The method of claim 74, wherein the probe comprises a sequence-specific binding protein.
- 76. The method of claim 75, wherein the probe comprises at least one Zn2+ finger protein.
- 77. The method of claim 75, wherein the probe comprises a plurality of Zn2+ finger proteins.
- 78. The method of claim 75, wherein the probe comprises at least two Zn2+ finger proteins, and wherein the each of the Zn2+ finger proteins comprises a moiety that promotes cooperative binding to DNA.
- 79. The method of claim 75, wherein the probe comprises at least one dimer of two Zn2+ finger proteins.
- 80. The method of claim 75, wherein the probe comprises at least one oligonucleotide.
- 81. The method of claim 80, wherein the oligonucleotide is a genome-specific oligonucleotide.
- 82. The method of claim 80, wherein the oligonucleotide is an allele-specific oligonucleotide.
- 83. The method of claim 80, wherein the probe comprises a set of universal oligonucleotides.
- 84. The method of claim 80, wherein the oligonucleotide is an mRNA specific oligonucleotide.
- 85. The method of claim 84, wherein the oligonucleotide comprises a nucleotide sequence complementary to an mRNA splice junction.
- 86. The method of claim 1, 30 or 53, whereby the detector is an electrode, and electron current is detected as the monomers traverse the electrode.
- 87. The method of claim 86, wherein the defined volume of the substrate is a groove on the substrate surface, and the detector is at a position along the groove such that it locally contacts the nucleic acid as it traverses the groove.
- 88. The method of claim 1, 30 or 53, wherein the defined volume comprises an aperture located in the substrate, wherein the aperture includes an entry port and an exit port defining a channel there between.
- 89. The method of claim 88, wherein nucleic acid molecule interactions with the aperture are detected as electronic currents at first and second electrodes adjacent to the aperture and in electrical communication with said channel.
- 90. The method of claim 89, wherein current along the length of the channel is detected.
- 91. The method of claim 1, 30 or 53, wherein nucleic acid molecule interactions with the aperture are detected by measuring ionic conductance in the channel.
- 92. The method of claim 1, 30 or 53, wherein the nucleic acid molecule is caused to traverse the aperture by application of a voltage gradient.
- 93. The method of claim 1, 30 or 53, wherein the amplitude or duration of individual conductance measurements is indicative of sequential identity of monomers of the polymer molecule.
- 94. The method of claim 1, 30 or 53, wherein the number of changes in the conductance measurement is an indication of the number of modifications of the cross-sectional volume in the nucleic acid molecule.
- 95. The method of claim 1, 30 or 53, wherein the duration of the individual conductance measurement is an indication of the number of nucleotides in the polymer molecule.
- 96. A method for characterizing a nucleic acid molecule, comprising:
providing a nucleic acid molecule for characterization; modifying an electronic property of the nucleic acid molecule, wherein at least one nucleotide is modified; contacting the modified nucleic acid with a substrate, the substrate including a detector capable of identifying an electronic property of the nucleic acid molecule and the detector being responsive to modification of the electronic property of the nucleic acid molecule; causing the modified nucleic acid molecule to traverse a defined volume on or within the substrate, so that individual nucleotides of the modified nucleic acid molecule interact with the detector in sequential order, whereby data correlating with the modification of the electronic property of the nucleic acid molecule are obtained.
- 97. The method of claim 96, wherein the detector identifies a current tunneling property of the nucleic acid molecule.
- 98. A method for characterizing a nucleic acid molecule, the method comprising determining a property of the nucleic acid molecule selected from the group consisting of: determining the presence or absence of a single nucleic acid sequence in the nucleic acid molecule, determining the presence or absence of a set of nucleic acid sequences in the nucleic acid molecule, determining the distances between individual sequences in a set of nucleic acid sequences in the nucleic acid molecule, and determining the frequency at which a nucleic acid sequence is present in a nucleic acid molecule, wherein the nucleic acid sequence or set of nucleic acid sequences is present on a single contiguous nucleic acid molecule.
RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional Application Serial No. 60/299,878, filed Jun. 21, 2001, the entire contents of which are incorporated herein.
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] This invention was made, in part, with United States Government support under DARPA grant number N65236-98-1-5407. The Government has certain rights in this invention.
Provisional Applications (1)
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Number |
Date |
Country |
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60299878 |
Jun 2001 |
US |