Claims
- 1. A method for analyzing a nucleic acid molecule, comprising:
exposing a nucleic acid molecule to a sequence specific nicking enzyme, allowing the sequence specific nicking enzyme to introduce nicks into the nucleic acid molecule, exposing the nucleic acid molecule to a polymerase enzyme and labeled nucleotides, allowing the polymerase enzyme to incorporate labeled nucleotides into the nucleic acid molecule, and detecting a signal from the labeled nucleotides incorporated into the nucleic acid molecule.
- 2. The method of claim 1, wherein the signal is detected using a linear polymer analysis system.
- 3. The method of claim 2, wherein the linear polymer analysis system is a single molecule detection system.
- 4. The method of claim 2, wherein the linear polymer analysis system is selected from the group consisting of a Gene Engine™ system, an optical mapping system, and a DNA combing system.
- 5. The method of claim 1, wherein the nucleic acid molecule is genomic DNA.
- 6. The method of claim 1, wherein the nucleic acid is a non in vitro amplified nucleic acid molecule.
- 7. The method of claim 1, wherein the nucleic acid molecule is a single nucleic acid molecule.
- 8. The method of claim 1, wherein the nucleic acid molecule is exposed to a station to produce the signal from the labeled nucleotides incorporated into the nucleic acid molecule.
- 9. The method of claim 1, wherein the labeled nucleotide comprises a label selected from the group consisting of a fluorescent molecule, a chemiluminescent molecule, a radioisotope, an enzyme substrate, a biotin molecule, an avidin molecule, an electrical charged transducing molecule, a nuclear magnetic resonance molecule, a semiconductor nanocrystal, an electromagnetic molecule, an electrically conducting particle, a ligand, a microbead, a chromogenic substrate, an affinity molecule, a Qdot, a protein, a peptide, a nucleic acid, a carbohydrate, an antibody, an antibody fragment, an antigen, a hapten, and a lipid.
- 10. The method of claim 1, wherein the detection system is selected from the group consisting of a fluorescent detection system, an electrical detection system, a photographic film detection system, a chemiluminescent detection system, an enzyme detection system, an atom force microscopy (AFM) detection system, a scanning tunneling microscopy (STM) detection system, an optical detection system, a nuclear magnetic resonance (NMR) detection system, a near field detection system, a total internal reflection (TIR) system, and a electromagnetic detection system.
- 11. The method of claim 1, further comprising labeling the nucleic acid molecule with a backbone label.
- 12. The method of claim 1, wherein the polymerase enzyme is DNA polymerase I.
- 13. The method of claim 1, wherein the sequence specific nicking enzyme is selected from the group consisting of restriction endonucleases, modified restriction endonucleases, recombination enzymes, recombinase, transposases, engineered protein chimera, DNA repair enzymes including mismatch repair enzymes, helicases, topoisomerases, DNases, modified DNases, homing endonucleases, and synthetic restriction enzymes.
- 14. A method for analyzing a nucleic acid molecule, comprising:
determining a nicking pattern of a nucleic acid molecule in a biological sample from a subject, and comparing the nicking pattern of the nucleic acid molecule to a control.
- 15. The method of claim 14, further comprising determining a difference in the nicking pattern of the nucleic acid molecule as compared to a control.
- 16. The method of claim 15, wherein a difference in the nicking pattern of the nucleic acid molecule as compared to the control identifies a subject having or at risk of developing a disorder characterized by abnormal nicking of a nucleic acid molecule.
- 17. The method of claim 16, wherein the subject is a human.
- 18. The method of claim 14, wherein the nucleic acid molecule is genomic DNA.
- 19. The method of claim 16, wherein the subject has been exposed to a DNA damaging agent.
- 20. The method of claim 14, wherein the control is a normal cell.
- 21. The method of claim 14, wherein the control is a set of data from normal cells.
- 22. The method of claim 16, wherein the difference in the nicking pattern is an increase in a total level of nicking.
- 23. The method of claim 16, wherein the difference in the nicking pattern is a decrease in a total level of nicking.
- 24. The method of claim 16, wherein the difference in the nicking pattern is a difference in the location of nicking.
- 25. The method of claim 16, wherein the disorder is cancer.
- 26. The method of claim 25, wherein the cancer is breast cancer.
- 27. The method of claim 16, wherein the disorder is a DNA repair deficiency disorder.
- 28. The method of claim 14, wherein the nucleic acid molecule is a non in vitro amplified nucleic acid molecule.
- 29. The method of claim 14, wherein the nucleic acid molecule is nicked in vivo.
- 30. The method of claim 29, wherein the nicking pattern is determined by
exposing the nucleic acid molecule to a polymerase enzyme and labeled nucleotides, allowing the polymerase enzyme to incorporate labeled nucleotides into the nucleic acid molecule, and detecting a signal from the labeled nucleotides incorporated into the nucleic acid molecule.
- 31. The method of claim 30, wherein the polymerase enzyme is DNA polymerase I.
- 32. A method for screening a compound for the ability to damage a nucleic acid molecule, comprising
determining a nicking pattern in a nucleic acid molecule prior to and after exposure of the nucleic acid molecule to a compound, and comparing the nicking pattern prior to and after exposure of the nucleic acid molecule to the compound, wherein the nicking patterns are determined by
exposing the nucleic acid molecule to a polymerase enzyme and labeled nucleotides, allowing the polymerase enzyme to incorporate labeled nucleotides into the nucleic acid molecule, and detecting a signal from the labeled nucleotides incorporated into the nucleic acid molecule.
- 33. A method for assessing the efficacy of a therapeutic treatment, comprising:
determining a nicking pattern of nucleic acid molecule from a biological sample from a subject prior to and after the therapeutic treatment, and comparing the nicking pattern prior to the therapeutic treatment with the nicking pattern after the therapeutic treatment, wherein a difference in the nicking pattern as a result of the therapeutic treatment is an indicator of the efficacy of the therapeutic treatment.
- 34. The method of claim 33, wherein the difference in the nicking pattern is an increase in a total level of nucleic acid nicking.
- 35. The method of claim 33, wherein the difference in the nicking pattern is a decrease in a total level of nucleic acid nicking.
- 36. The method of claim 33, wherein the therapeutic treatment is an anti-cancer agent.
- 37. The method of claim 36, wherein the anti-cancer agent is a DNA damaging agent.
- 38. The method of claim 33, wherein the nicking pattern is determined by
exposing the nucleic acid molecule to a polymerase enzyme and labeled nucleotides, allowing the polymerase enzyme to incorporate labeled nucleotides into the nucleic acid molecule, and detecting a signal from the labeled nucleotides incorporated into the nucleic acid molecule.
- 39. A system for optically analyzing a nucleic acid molecule comprising:
an optical source for emitting optical radiation of a known wavelength; an interaction station for receiving the optical radiation in an optical path and for receiving the nucleic acid molecule that is exposed to the optical radiation to produce detectable signals; dichroic reflectors in the optical path for creating at least two separate wavelength bands of the detectable signals; optical detectors constructed to detect radiation including the signals resulting from interaction of the nucleic acid molecule with the optical radiation; and a processor constructed and arranged to analyze the nucleic acid molecule based on the detected radiation including the signals, wherein the nucleic acid molecule is labeled using nick translation.
- 40. The system of claim 39, wherein the nucleic acid molecule is labeled with a label selected from the group consisting of a fluorescent molecule, a chemiluminescent molecule, a radioisotope, an enzyme substrate, a biotin molecule, an avidin molecule, an electrical charge transducing molecule, a nuclear magnetic resonance molecule, a semiconductor nanocrystal, an electromagnetic molecule, an electrically conducting microparticle, a protein, a peptide, an antibody, an antigen, an antibody fragment, a hapten, a ligand, a Qdot and a microbead.
- 41. The method of claim 39, wherein the nucleic acid molecule is a non in vitro amplified nucleic acid molecule.
- 42. The method of claim 39, wherein the nucleic acid molecule is genomic DNA.
- 43. The system of claim 39, wherein the interaction station includes a slit having a slit width in the range of 1 nm to 500 nm and producing a localized radiation spot.
- 44. The system of claim 43, wherein the slit width is in the range of 10 nm to 100 nm.
- 45. The system of claim 43, wherein further comprising a microchannel arranged with the slit to produce the localized radiation spot, the microchannel being constructed to receive and advance the polymer units through the localized radiation spot.
- 46. The system of claim 45, further comprising a polarizer, wherein the optical source includes a laser constructed to emit a beam of radiation and the polarizer is arranged to polarize the beam prior to reaching the slit.
- 47. The system of claim 46, wherein the polarizer is arranged to polarize the beam in parallel to the width of the slit.
- 48. The method of claim 39, wherein the nucleic acid molecule is labeled using nick translation comprising
exposing the nucleic acid molecule to a polymerase enzyme and labeled nucleotides, and allowing the polymerase enzyme to incorporate labeled nucleotides into the nucleic acid molecule.
- 49. The method of claim 48, further comprising
exposing the nucleic acid molecule to a sequence specific nicking enzyme, allowing the sequence specific nicking enzyme to introduce nicks into the nucleic acid molecule, prior to exposing the nucleic acid molecule to the polymerase enzyme.
- 50. The method of claim 48, wherein the labeled nucleotides are detected using a detection system selected from the group consisting of a fluorescent detection system, an electrical detection system, a photographic film detection system, a chemiluminescent detection system, an enzyme detection system, an atom force microscopy (AFM) detection system, a scanning tunneling microscopy (STM) detection system, an optical detection system, a nuclear magnetic resonance (NMR) detection system, a near field detection system, a total internal reflection (TIR) system, and a electromagnetic detection system.
- 51. A method for analyzing a nucleic acid molecule comprising:
generating optical radiation of a known wavelength to produce a localized radiation spot; passing a labeled nucleic acid molecule through a microchannel; irradiating the labeled nucleic acid molecule at the localized radiation spot; sequentially detecting radiation resulting from interaction of the labeled nucleic acid with the optical radiation at the localized radiation spot; and analyzing the labeled nucleic acid molecule based on the detected radiation, wherein the nucleic acid molecule is labeled using nick translation.
- 52. The method of claim 51, further comprising employing an electric field to pass the nucleic acid molecule through the microchannel.
- 53. The method of claim 51, wherein the detecting includes collecting the signals over time while the nucleic acid molecule is passing through the microchannel.
- 54. The method of claim 50, wherein the nucleic acid molecule is labeled using a nick translation approach comprising
exposing the nucleic acid molecule to a polymerase enzyme and labeled nucleotides, and allowing the polymerase enzyme to incorporate labeled nucleotides into the nucleic acid molecule.
- 55. The method of claim 54, further comprising
exposing the nucleic acid molecule to a sequence specific nicking enzyme, allowing the sequence specific nicking enzyme to introduce nicks into the nucleic acid molecule, prior to exposing the nucleic acid molecule to the polymerase enzyme.
- 56. The method of claim 54, wherein the labeled nucleotides are detected using a detection system selected from the group consisting of a fluorescent detection system, an electrical detection system, a photographic film detection system, a chemiluminescent detection system, an enzyme detection system, an atom force microscopy (AFM) detection system, a scanning tunneling microscopy (STM) detection system, an optical detection system, a nuclear magnetic resonance (NMR) detection system, a near field detection system, a total internal reflection (TIR) system, and a electromagnetic detection system.
- 57. The method of claim 54, wherein the labeled nucleotides are conjugated to a label selected from the group consisting of a fluorescent molecule, a chemiluminescent molecule, a radioisotope, an enzyme substrate, a biotin molecule, an avidin molecule, a Qdot, an electrical charge transducing molecule, a nuclear magnetic resonance molecule, a semiconductor nanocrystal, an electromagnetic molecule, a protein, a peptide, an antibody, an antibody fragment, an antigen, a hapten, a ligand, and a microbead.
- 58. The method of claim 51, wherein the nucleic acid molecule is a non in vitro amplified nucleic acid molecule.
- 59. The method of claim 51, wherein the nucleic acid molecule is genomic DNA.
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent Application filed Jun. 8, 2001, entitled “METHODS AND PRODUCTS FOR ANALYZING NUCLEIC ACIDS USING NICK TRANSLATION”, Ser. No. 60/297,080, the contents of which are incorporated by reference herein in their entirety.
Provisional Applications (1)
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Number |
Date |
Country |
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60297080 |
Jun 2001 |
US |