Methods of analyzing polymers using a spatial network of fluorophores and fluorescence resonance energy transfer

• US 6,263,286 B1
• Filed: 08/13/1999
• Issued: 07/17/2001
• Est. Priority Date: 08/13/1998
• Status: Expired due to Term

• Current Assignee: U.S. Genomics, Inc (Toxic Reports LLC)
• Original Assignee: U.S. Genomics, Inc (Toxic Reports LLC)
• Inventors: Chan, Eugene Y., Gilmanshin, Rudolf
• Primary Examiner(s): Brusca, John S.
• Assistant Examiner(s): Lundgren, Jeffrey S.
• Application Number:
  US09/374,902
• Time in Patent Office: 704 Days
• Field of Search: 435/6, 702/19
• US Class Current: 702/19
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/0663 Stretching or orienting elongated molecules or particles
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/086 using baffles or other fixed flow obstructions
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: G01N 33/48 Biological material, e.g. blood, urine G01N33/02, G01N33/26, G01N33/44, G01N33/46 take precedence; Haemocytometers counting blood corpuscules distributed over a surface by scanning the surface G06M11/02

Abstract

The present invention relates to methods and apparatuses for analyzing molecules, particularly polymers, and molecular complexes with extended or rod-like conformations. In particular, the methods and apparatuses are used to identify repetitive information in molecules or molecular ensembles, which is interpreted using an autocorrelation function in order to determine structural information about the molecules. The methods and apparatuses of the invention are used for, inter alia, determining the sequence of a nucleic acid, determining the degree of identity of two polymers, determining the spatial separation of specific sites within a polymer, determining the length of a polymer, and determining the velocity with which a molecule penetrates a biological membrane.

Claims

1. A method for analyzing extended objects comprising:

(a) moving with respect to at least a first station a plurality of similar extended objects that are each similarly labeled at similar positions with at least a first unit-specific marker and a second unit-specific marker to generate a plurality of object-dependent impulses as the similar extended objects pass the first station, wherein the first and second unit-specific markers are at different positions along each similar extended object, wherein the similar extended objects are extended molecules or extended molecular complexes;

(b) measuring the generated plurality of object-dependent impulses as a function of time, wherein the object-dependent impulses generated due to said first unit-specific marker of an individual one of said plurality of similar extended objects passing said first station and said object-dependent impulses generated due to said second unit-specific marker of said individual one of said plurality of similar extended objects passing said first station are resolved in time; and

(c) calculating an autocorrelation function of said measured plurality of object-dependent impulses, to analyze the extended objects.

2. The method of claim 1 wherein said plurality of similar extended objects are moved with respect to the first station at similar velocities.

3. The method of claim 1, wherein the plurality of similar extended objects is a plurality of polymers.

4. The method of claim 3, wherein each polymer in the plurality of polymers is identical to every other polymer in the plurality of polymers.

5. The method of claim 4, wherein each polymer in the plurality of polymers is labeled with identical unit-specific markers at identical positions within the polymer.

6. The method of claim 3, 4 or 5, wherein each polymer in the plurality of polymers is a nucleic acid.

7. The method of claim 6, wherein the nucleic acid is DNA.

8. The method of claim 1, wherein the autocorrelation function is defined by the formula:

$G<br><br>\left(\mathrm{\tau <br><br>}\right)=1/T<br><br>{\int <br><br>}_0^T<br><br>I<br><br>\left(t\right)<br><br>I<br><br>\left(t+\mathrm{\tau <br><br>}\right)<br><br>\text{\hspace{1em}<br><br>}<br><br><br><br>t$

where G(τ

) is the autocorrelation function of the time dependence of measured object-dependent impulses, T is the total time of measurement of I(t), and I(t) is the object-dependent impulse measurement at each time point t.

9. The method of claim 1, wherein the autocorrelation function is defined by the formula:

$G_j=\left(1/N\right)<br><br>\underset{i=0}{\overset{N}{\sum <br><br>}}<br><br>I_i<br><br>I_{i+j}$

where G_j is the autocorrelation function of the time dependence of measured object-dependent impulses at time jΔ

t, N is the total number of data values, I_i is the object-dependent impulse measurement at time t_i, I_i+j is the object-dependent impulse measurement at time t_i+jΔ

t, and Δ

t is a time interval.

10. The method of claim 1, wherein the at least a first station is a plurality of stations.

11. The method of claim 10, wherein the plurality of similar extended objects is moved through a lattice of beads and the plurality of stations is positioned on a subset of the plurality of beads.

12. The method of claim 8, 9 or 11, wherein the plurality of similar extended objects is a plurality of polymers.

13. The method of claim 12, wherein each polymer in the plurality of polymers is a nucleic acid.

14. The method of claim 13, wherein the nucleic acid is DNA.

15. The method of claim 10, wherein the plurality of similar extended objects is moved through a channel, said channel having a first end, a second end, and at least one wall, the plurality of stations being positioned along the at least one wall.

16. The method of claim 10, wherein the plurality of similar extended objects is moved through a channel, said channel having a first end, a second end, and at least one wall, the plurality of stations being positioned at said first end or said second end.

17. The method of claim 15 or 16, wherein the plurality of similar extended objects is a plurality of polymers.

18. The method of claim 17, wherein each polymer in the plurality of polymers is a nucleic acid.

19. The method of claim 18, wherein the nucleic acid is DNA.

20. The method of claim 10, wherein the plurality of similar extended objects is moved simultaneously through a plurality of channels, each channel in said plurality of channels having a first end, a second end, and at least one wall, each channel having at least one station positioned along the at least one wall.

21. The method of claim 10, wherein the plurality of similar extended objects is moved simultaneously through a plurality of channels, each channel in said plurality of channels having a first end, a second end, and at least one wall, each channel having at least one station positioned at said first end or said second end.

22. The method of claim 20 or 21, wherein the plurality of similiar extended objects is a plurality of polymers.

23. The method of claim 22, wherein each polymer in the plurality of polymers is a nucleic acid.

24. The method of claim 23, wherein the nucleic acid is DNA.

25. The method of claim 1, wherein the plurality of similar extended objects is moved through the action of at least one molecular motor.

26. The method of claim 25, wherein the at least one molecular motor is a plurality of molecular motors in solution.

27. The method of claim 25 or 26, wherein the plurality of similar extended objects is a plurality of polymers.

28. The method of claim 27, wherein each polymer in the plurality of polymers is a nucleic acid.

29. The method of claim 28, wherein the nucleic acid is DNA.

30. The method of claim 1, wherein the object-dependent impulses generated due to said first unit-specific marker of an individual one of said plurality of similar extended objects passing said first station and said object-dependent impulses generated due to said second unit-specific marker of said individual one of said plurality of similar extended objects passing said first station are different.

31. The method of claim 30, wherein the plurality of similar extended objects is a plurality of polymers.

32. The method of claim 31, wherein each polymer in the plurality of polymers is a nucleic acid.

33. The method of claim 32, wherein the nucleic acid is DNA.

34. The method of claim 1 wherein the object-dependent impulses are fluorescence resonance energy transfer.

35. The method of claim 34, wherein said first station comprises at least one donor fluorophore and said first unit-specific marker and said second unit-specific marker each comprise at least one acceptor fluorophore.

36. The method of claim 34, wherein said first station comprises at least one acceptor fluorophore and said first unit-specific marker and said second unit-specific marker each comprise at least one donor fluorophore.

37. The method of claim 34, 35 or 36, wherein the plurality of similar extended objects is a plurality of polymers.

38. The method of claim 37, wherein each polymer in the plurality of polymers is a nucleic acid.

39. The method of claim 38, wherein the nucleic acid is DNA.

40. The method of claim 1, wherein repetitive information in the measured plurality of object-dependent impulses provides information about the length of the extended objects.

41. The method of claim 1, wherein analysis of the extended objects provides information about the distance between said first unit-specific marker and said second unit-specific marker on the extended objects.

42. The method of claim 2, wherein analysis of the extended objects provides information about the velocity of the extended objects.

43. The method of claim 1, wherein analysis of the extended objects provides information about the linear arrangement of units within the extended objects.

44. The method of claim 40, 41, 42 or 43, wherein the plurality of extended objects is a plurality of polymers.

45. The method of claim 44, wherein each polymer in the plurality of polymers is a nucleic acid.

46. The method of claim 45, wherein the nucleic acid is DNA.

47. A method for analyzing extended objects comprising calculating an autocorrelation function of measured object-dependent impulses, wherein said object dependent impulses have been produced by:

(a) moving with respect to at least a first station a plurality of similar extended objects that are each similarly labeled at similar positions with at least a first unit-specific marker and a second unit-specific marker to generate a plurality of object-dependent impulses as the similar extended objects pass the first station, wherein the first and second unit-specific markers are at different positions along each similar extended object, wherein the similar extended objects are extended molecules or extended molecular complexes; and

(b) measuring the generated plurality of object-dependent impulses as a function of time, wherein the object-dependent impulses generated due to said first unit-specific marker of an individual one of said plurality of similar extended objects passing said first station and said object-dependent impulses generated due to said second unit-specific marker of said individual one of said plurality of similar extended objects passing said first station are resolved in time.

48. The method of claim 47, wherein the extended objects are polymers.

49. The method of claim 48 wherein the polymers are nucleic acids.

50. The method of claim 49, wherein the nucleic acids are DNA.

51. The method of claim 47, wherein the autocorrelation function is defined by the formula:

$G<br><br>\left(\mathrm{\tau <br><br>}\right)=1/T<br><br>{\int <br><br>}_0^T<br><br>I<br><br>\left(t\right)<br><br>I<br><br>\left(t+\mathrm{\tau <br><br>}\right)<br><br>\text{\hspace{1em}<br><br>}<br><br><br><br>t$

where G(τ

) is the autocorrelation function of the time dependence of measured object-dependent impulses, T is the total time of measurement of I(t), and I(t) is the object-dependent impulse measurement at each time point t.

52. The method of claim 47, wherein the autocorrelation function is defined by the formula:

$G_j=\left(1/N\right)<br><br>\underset{i=0}{\overset{N}{\sum <br><br>}}<br><br>I_i<br><br>I_{i+j}$

where G_j is the autocorrelation function of the time dependence of measured object-dependent impulses at time jΔ

t, N is the total number of data values, I_i is the object-dependent impulse measurement at time t_i, I_i+j is the object-dependent impulse measurement at time t_i+jΔ

t, and Δ

t is a time interval.

53. The method of claim 47, wherein the object-dependent impulses generated due to said first unit-specific marker of an individual one of said plurality of similar extended objects passing said first station and said object-dependent impulses generated due to said second unit-specific marker of said individual one of said plurality of similar extended objects passing said first station are different.

54. The method of claim 53, wherein the extended objects are polymers.

55. The method of claim 51, 52 or 54, wherein the polymers are nucleic acids.

56. The method of claim 55, wherein the nucleic acids are DNA.

57. The method of claim 47 wherein the object-dependent impulses are fluorescence resonance energy transfer.

58. The method of claim 57, wherein the extended objects are polymers.

59. The method of claim 58, wherein the polymers are nucleic acids.

60. The method of claim 59, wherein the nucleic acid is DNA.

61. The method of claim 47, wherein the analysis of the extended objects provides information about the length of the extended objects.

62. The method of claim 47, wherein analysis of the extended objects provides information about the distance between said first unit-specific marker and said second unit-specific marker on the extended objects.

63. The method of claim 47, wherein analysis of the extended objects provides information about the velocity of the extended objects.

64. The method of claim 47, wherein analysis of the extended objects provides information about the linear arrangement of units within the extended objects.

65. The method of claim 61, 62, 63 or 64, wherein the extended objects are polymers.

66. The method of claim 65, wherein the polymers are nucleic acids.

67. The method of claim 66, wherein the nucleic acids are DNA.