Differential tagging of polymers for high resolution linear analysis

• US 20030059822A1
• Filed: 09/18/2002
• Published: 03/27/2003
• Est. Priority Date: 09/18/2001
• Status: Abandoned Application

• Current Assignee: U.S. Genomics, Inc (Toxic Reports LLC)
• Original Assignee: U.S. Genomics, Inc (Toxic Reports LLC)
• Inventors: Chan, Eugene Y., Gilmanshin, Rudolf, Fuchs, Martin
• Application Number:
  US10/246,779
• Publication Number:
  US 20030059822A1
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

The invention provides methods and systems for improved spatial resolution of signal detection, particularly as applied to the analysis of polymers such as biological polymers. The methods and systems comprise differentially tagging polymers in order to increase resolution.

Claims

1. A method for analyzing a polymer comprising:

a) providing a detection station having a known detection resolution;

b) labeling the polymer with first and second unit specific markers, the first unit specific marker including a first label and the second unit specific marker including a second label distinct from the first label, wherein the first and second unit specific markers are spaced apart on the polymer such that, if the labels were not distinct from each other, they would be separated by a distance less than the detection resolution;

c) exposing the polymer labeled as in (b) to the detection station to produce distinct first and second signals arising from the first and second labels; and

d) identifying the distinct first and second signals.

2. The method of claim 1, wherein the first unit specific marker is different from the second unit specific marker.

3. The method of claim 1, wherein the first unit specific marker is identical to the second unit specific marker.

4. The method of claim 1, wherein the first unit specific marker and the second unit specific marker are positioned immediately adjacent to one another.

5. The method of claim 1, wherein the first unit specific marker and the second unit specific marker are spatially separated from one another by at least two units.

6. The method of claim 1, wherein the polymer is labeled with a third unit specific marker comprising a third label.

7. The method of claim 6, wherein the third unit specific marker is spaced apart from the first and second unit specific markers by a distance greater than the known detection resolution.

8. The method of claim 1, wherein the first and second unit specific markers are nucleic acid molecules.

9. The method of claim 1, wherein the first and second unit specific markers are peptide nucleic acid molecules or locked nucleic acid molecules.

10. The method of claim 8, wherein the first and second unit specific markers have an identical nucleotide sequence.

11. The method of claim 8, wherein the first and second unit specific markers are less than 12 bases in length.

12. The method of claim 8, wherein the first and second unit specific markers are at least 4 bases in length.

13. The method of claim 1, wherein the first label and second label are independently selected from the group consisting of an electron spin resonance molecule, a fluorescent molecule, a chemiluminescent molecule, a radioisotope, an enzyme substrate, an enzyme, a biotin molecule, an avidin molecule, an electrical charge transferring molecule, a semiconductor nanocrystal, a semiconductor nanoparticle, a colloid gold nanocrystal, a ligand, a microbead, a magnetic bead, a paramagnetic molecule, a quantum dot, a chromogenic substrate, an affinity molecule, a protein, a peptide, a nucleic acid, a carbohydrate, a hapten, an antigen, an antibody, an antibody fragment, and a lipid.

14. The method of claim 1, wherein the signals are detected using a detection system selected from the group consisting of an electron spin resonance (ESR) detection system, a charge coupled device (CCD) detection system, a fluorescent detection system, an electrical detection system, an electromagnetic detection system, a photographic film detection system, a chemiluminescent detection system, an enzyme detection system, an atomic 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, and a total internal reflection (TIR) detection system.

15. The method of claim 1, wherein the polymer is a nucleic acid molecule.

16. The method of claim 1, wherein the polymer is genomic DNA.

17. The method of claim 1, wherein the polymer comprises a backbone that includes a label.

18. A system for optically analyzing a polymer of linked units comprising:

a) an optical source for emitting optical radiation of a known wavelength;

b) an interaction station for receiving the optical radiation in an optical path and for sequentially receiving units of the polymer that are exposed to the optical radiation to produce detectable signals;

c) dichroic reflectors in the optical path for creating at least two separate wavelength bands of the detectable signals;

d) optical detectors constructed to detect radiation including the signals resulting from interaction of the units with the optical radiation; and

e) a processor constructed and arranged to analyze the polymer based on the detected radiation including the signals.

19. The system of claim 18, wherein the units of the polymer are labeled with at least two radiation sensitive labels.

20. The system of claim 18, wherein the interaction station includes a slit having a slit width in the range of 1 nm to 500 nm, the slit producing a localized radiation spot.

21. The system of claim 20, wherein the slit width is in the range of 10 nm to 100 nm.

22. The system of claim 18, wherein the interaction station includes a microchannel and a slit having a submicron width arranged to produce the localized radiation spot, the microchannel being constructed to receive and advance the polymer units through the localized radiation spot.

23. The system of claim 21, further including a polarizer and wherein the optical source includes a laser constructed to emit a beam of radiation, the polarizer being arranged to polarize the beam prior to reaching the slit.

24. The system of claim 21, wherein the polarizer is arranged to polarize the beam in parallel to the width of the slit.

25. A method for analyzing a polymer of linked units comprising:

a) providing a microchannel;

b) generating optical radiation of a known wavelength to produce a localized radiation spot at the microchannel to define a detection station having a known detection resolution;

c) labeling the polymer with first and second unit specific markers, the first unit specific marker including a first label and the second unit specific marker including a second label distinct from the first label, wherein the markers are spaced apart on the polymer such that, if the labels were not distinct from each other, they would be separated by a distance less than the detection resolution;

d) sequentially exposing the first and second labels to the localized radiation spot;

e) sequentially detecting radiation of at least two distinct wavelength bands resulting from interaction of the first and second labels with the localized radiation spot; and

f) analyzing the polymer using the detected wavelength bands.

26. The method of claim 25, further comprising applying an electric field to move the polymer through the microchannel.

27. The method of claim 25, further comprising applying pressure to move the polymer through the microchannel.

28. The method of claim 25, further comprising applying suction to move the polymer through the microchannel.

29. The method of claim 25, wherein the first and second labels are independently selected from the group consisting of an electron spin resonance molecule, a fluorescent molecule, a chemiluminescent molecule, a radioisotope, an enzyme substrate, an enzyme, a biotin molecule, an avidin molecule, an electrical charge transferring molecule, a semiconductor nanocrystal, a semiconductor nanoparticle, a colloid gold nanocrystal, a ligand, a microbead, a magnetic bead, a paramagnetic molecule, a quantum dot, a chromogenic substrate, an affinity molecule, a protein, a peptide, a nucleic acid, a carbohydrate, a hapten, an antigen, an antibody, an antibody fragment, and a lipid.

30. The method of claim 25, wherein the first and second labels are fluorophores.

31. The method of claim 25, wherein the detecting includes collecting the first and second signals arising from the first and second labels while the first and second unit specific markers are moving through the microchannel.

32. The method of claim 25, wherein the first unit specific marker is different from the second unit specific marker.

33. The method of claim 25, wherein the first unit specific marker is identical to the second unit specific marker.

34. The method of claim 25, wherein the first unit specific marker and the second unit specific marker are positioned immediately adjacent to one another.

35. The method of claim 25, wherein the first unit specific marker and the second unit specific marker are spatially separated from one another by at least two units.

36. The method of claim 25, wherein the polymer is labeled with a third unit specific marker, including a third label.

37. The method of claim 36, wherein the third unit specific marker is spaced apart from the first and second unit specific markers by a distance greater than the minimum detection resolution.

38. The method of claim 25, wherein the first and second unit specific markers are nucleic acid molecules.

39. The method of claim 25, wherein the first and second unit specific markers are peptide nucleic acid molecules or locked nucleic acid molecules.

40. The method of claim 38, wherein the first and second unit specific markers have an identical nucleotide sequence.

41. The method of claim 38, wherein the first and second unit specific markers are less than 12 bases in length.

42. The method of claim 38, wherein the first and second unit specific markers are at least 4 bases in length.

43. The method of claim 25, wherein the first label and second label are independently selected from the group consisting of an electron spin resonance molecule, a fluorescent molecule, a chemiluminescent molecule, a radioisotope, an enzyme substrate, an enzyme, a biotin molecule, an avidin molecule, an electrical charge transferring molecule, a semiconductor nanocrystal, a semiconductor nanoparticle, a colloid gold nanocrystal, a ligand, a microbead, a magnetic bead, a paramagnetic molecule, a quantum dot, a chromogenic substrate, an affinity molecule, a protein, a peptide, nucleic acid, a carbohydrate, a hapten, an antigen, an antibody, an antibody fragment, and a lipid.

44. The method of claim 25, wherein the signals are detected using a detection system selected from the group consisting of an electron spin resonance (ESR) detection system, a charge coupled device (CCD) detection system, a fluorescent detection system, an electrical detection system, an electromagnetic detection system, a photographic film detection system, a chemiluminescence detection system, an enzyme detection system, an atomic 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, and a total internal reflection (TIR) detection system.

45. The method of claim 25, wherein the polymer is a nucleic acid molecule.

46. The method of claim 25, wherein the polymer is genomic DNA.

47. The method of claim 25, wherein the polymer comprises a backbone that includes a label.

48. A method for analyzing a polymer comprising

labeling a polymer with a set of unit specific markers, wherein each unit specific marker of the set recognizes and binds to units of identical sequence within the polymer and wherein each unit specific marker is labeled with one of at least two distinct labels, and

detecting signals arising from the labels to analyze the polymer.

49. The method of claim 48, wherein about 50% of the unit specific markers are labeled with a first label and about 50% of the unit specific markers are labeled with a second label.

50. The method of claim 48, wherein each unit specific marker is labeled with one of at least three distinct labels.

51. The method of claim 48, wherein each unit specific marker is labeled with one of at least four distinct labels.

52. The method of claim 48, wherein the unit specific markers are nucleic acid molecules.

53. The method of claim 48, wherein the unit specific markers are peptide nucleic acid molecules or locked nucleic acid molecules.

54. The method of claim 52, wherein the unit specific markers have identical sequence.

55. The method of claim 52, wherein the unit specific markers are less than 12 bases in length.

56. The method of claim 48, wherein the labels are of a type selected from the group consisting of an electron spin resonance molecule, a fluorescent molecule, a chemiluminescent molecule, a radioisotope, an enzyme substrate, an enzyme, a biotin molecule, an avidin molecule, an electrical charge transferring molecule, a semiconductor nanocrystal, a semiconductor nanoparticle, a colloid gold nanocrystal, a ligand, a microbead, a magnetic bead, a paramagnetic molecule, a quantum dot, a chromogenic substrate, an affinity molecule, a protein, a peptide, a nucleic acid, a carbohydrate, a hapten, an antigen, an antibody, an antibody fragment, and a lipid.

57. The method of claim 48, wherein the distinct labels are of different types.