Single molecule arrays for genetic and chemical analysis

• US 20070099208A1
• Filed: 06/13/2006
• Published: 05/03/2007
• Est. Priority Date: 06/15/2005
• Status: Active Grant

• Current Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Original Assignee: Callida Genomics, Inc. (SBH Genomics, Inc.)
• Inventors: Drmanac, Radoje, Callow, Matthew, Yeung, George, Drmanac, Snezana, Hauser, Brian
• Granted Patent: US 8,445,194 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C07K 1/047 Simultaneous synthesis of different peptide species; Peptide libraries
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• 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 2521/307 Single strand endonuclease
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/313 Branched oligonucleotides
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: G01N 15/1404 Handling flow, e.g. hydrodynamic focusing
• CPC Class Codes: G01N 15/1434 Optical arrangements
• CPC Class Codes: Y10S 977/778 within specified host or matrix material, e.g. nanocomposite films
• CPC Class Codes: Y10S 977/789 in array format
• CPC Class Codes: Y10S 977/792 : Nucleic acid array, e.g. human genome array
• CPC Class Codes: Y10S 977/88 : with arrangement, process, or apparatus for testing
• CPC Class Codes: Y10S 977/882 : Assembling of separate components, e.g. by attaching

Abstract

Random arrays of single molecules are provided for carrying out large scale analyses, particularly of biomolecules, such as genomic DNA, cDNAs, proteins, and the like. In one aspect, arrays of the invention comprise concatemers of DNA fragments that are randomly disposed on a regular array of discrete spaced apart regions, such that substantially all such regions contain no more than a single concatemer. Preferably, such regions have areas substantially less than 1 μm² and have nearest neighbor distances that permit optical resolution of on the order of 10⁹ single molecules per cm². Many analytical chemistries can be applied to random arrays of the invention, including sequencing by hybridization chemistries, sequencing by synthesis chemistries, SNP detection chemistries, and the like, to greatly expand the scale and potential applications of such techniques.

Claims

1. An array of polymer molecules comprising:

a support having a surface; and

a plurality of polymer molecules attached to the surface, wherein each polymer molecule has a random coil state and comprises a branched or linear structure of multiple copies of one or more linear polymeric units, such that the polymer molecule is attached to the surface within a region substantially equivalent to a projection of the random coil on the surface and randomly disposed at a density such that at least thirty percent of the polymer molecules are separately detectable.

2. The array of polymer molecules of claim 1 wherein said one or more linear polymeric units are each single stranded polynucleotides and wherein said surface has reactive functionalities or capture oligonucleotides attached thereto and wherein said polymer molecules are each attached to said surface by one or more linkages formed by one or more reactive functionalities reacting with complementary functionalities of said polymers molecules or by one or more complexes formed between the capture oligonucleotides and complementary sequences of the polymer molecules.

3. The array of claim 2 wherein said surface is a planar surface having an array of discrete spaced apart regions, wherein each discrete spaced apart region has a size substantially equivalent to said projection of said random coil of said polymer molecule and contains said reactive functionalities or said capture oligonucleotides attached thereto.

4. The array of claim 3 wherein each of said discrete spaced apart regions has an area of less than 1 μ

m².

5. The array of claim 4 wherein said discrete spaced apart regions form a regular array with a nearest neighbor distance in the range of from 0.1 to 20 μ

m and wherein a majority of said discrete spaced apart regions contain no more than one said polymer molecules.

6. The array of claim 5 wherein said polymer molecules are randomly distributed on said discrete spaced apart regions and wherein said nearest neighbor distance is in the range of from 0.3 to 3 μ

m.

7. The array of claim 6 wherein each of said polymer molecules is a polynucleotide molecule comprising a concatemer of multiple copies of a target sequence and an adaptor oligonucleotide.

8. The array of claim 4 wherein said discrete spaced apart regions are wells in said support, the wells each having an opening with an area equal to or less than that of said discrete spaced apart regions.

9. An array of polynucleotide molecules comprising:

a support having a surface; and

a plurality of polynucleotide molecules attached to the surface, wherein each polynucleotide molecule has a random coil state and comprises a concatemer of multiple copies of a target sequence such that the polynucleotide molecule is attached to the surface within a region substantially equivalent to a projection of the random coil on the surface and randomly disposed at a density such that at least thirty percent of the polynucleotide molecules have a nearest neighbor distance of at least fifty nm.

10. The array of claim 9 wherein said surface has reactive functionalities attached thereto and wherein said polynucleotide molecules are each attached to said surface by one or more linkages formed by one or more reactive functionalities reacting with complementary functionalities of said polynucleotide molecules.

11. The array of claim 10 wherein said surface is a planar surface having an array of discrete spaced apart regions, wherein each discrete spaced apart region has a size substantially equivalent to said projection of said random coil of said polynucleotide molecule and contains said reactive functionalities attached thereto and wherein such regions have at most one of said polynucleotides attached.

12. The array of claim 11 wherein said reactive functionalities are hydrophobic functionalities.

13. The array of claim 11 wherein said discrete spaced apart region has an area of less than 1 μ

m².

14. The array of claim 13 wherein said discrete spaced apart regions are wells in said support, the wells each having an opening with an area equal to or less than that of said discrete spaced apart regions.

15. The array of claim 13 wherein said polynucleotides are randomly distributed on said discrete spaced apart regions and wherein said nearest neighbor distance is in the range of from 0.3 to 3 μ

m.

16. The array of claim 15 wherein substantially every said discrete spaced apart region has a polynucleotide attached.

17. The array of claim 15 wherein said concatemer comprises alternating copies of said target sequence and said adaptor oligonucleotide.

18. The array of claim 17 wherein each of said concatemers contains at least 10 copies of its respective target sequence.

19. The array of claim 17 wherein said target sequences each have a length in the range of from 50 to 500 nucleotides and wherein said adaptor oligonucleotide has a length in the range of from 6 to 60 nucleotides.

20. The array of claim 15 wherein all of said reactive functionalities are the same.

21. The array of claim 15 wherein each of said concatemers comprises at least one decoder oligonucleotide for identifying its respective target sequence.

22. The array of claim 15 wherein said discrete spaced apart regions are disposed on said planar surface in a rectilinear pattern wherein each discrete spaced apart region has an area enclosed by a circle having a diameter of from 0.1 to 10 μ

m.

23. The array of claim 11 wherein said discrete spaced apart regions form a regular array with a nearest neighbor distance in the range of from 0.1 to 20 μ

m.

24. The array of claim 23 wherein each of said discrete spaced apart regions is surrounded by an inter-regional space that is substantially free of said polynucleotides.

25. The array of claim 10 wherein said polynucleotides are randomly disposed on said planar surface such that at least a majority of said polynucleotides are optically resolvable.

26. The array of claim 25 wherein at least seventy percent of said polynucleotides are optically resolvable.

27. An array of single molecules comprising:

a support having a surface having a regular array of discrete spaced apart regions, wherein each discrete spaced apart region has an area of less than 1 μ

m² and contains reactive functionalities attached thereto; and

a plurality of single molecules attached to the surface, wherein each single molecule comprises a macromolecular structure and at least one analyte having an attachment moiety, such that each macromolecular structure comprises a unique functionality and a plurality of attachment functionalities that are capable of forming linkages with the reactive functionalities of the discrete spaced apart regions, and such that the analyte is attached to the macromolecular structure by a linkage between the unique functionality and the attachment moiety of the analyte;

wherein the plurality of single molecules are randomly disposed on the discrete spaced apart regions such that at least a majority of the discrete spaced apart regions contain only one single molecule.

28. The array of claim 27 wherein said macromolecular structure is a branched or linear polymer.

29. The array of claim 28 wherein said discrete spaced apart regions are wells in said support, the wells each having an opening with an area equal to or less than that of said discrete spaced apart regions.

30. The array of claim 28 wherein said discrete spaced apart regions each have a nearest neighbor distance in the range of from 0.1 to 20 μ

m and wherein said macromolecular structures are branched or linear polynucleotides.

31. The array of claim 30 wherein said nearest neighbor distance is in the range of from 0.3 to 3 μ

m.

32. The array of claim 31 wherein substantially every said discrete spaced apart region has a single molecule attached.

33. The array of claim 31 wherein said macromolecular structures are linear polynucleotides.

34. The array of claim 31 wherein said discrete spaced apart regions are disposed on said planar surface in a rectilinear pattern wherein each discrete spaced apart region has an area enclosed by a circle having a diameter of from 0.1 to 10 μ

m.

35. The array of claim 31 wherein said analyte is a polynucleotide or a protein.

36. The array of claim 31 wherein each of said discrete spaced apart regions is surrounded by an inter-regional space that is substantially free of said macromolecular structures.

37. The array of claim 31 wherein said unique functionality is a first oligonucleotide and wherein said attachment moiety is a second oligonucleotide having a sequence complementary to the first oligonucleotide.

38. The array of claim 37 wherein said analyte is a polynucleotide.

39. An array of polynucleotide molecules comprising:

a support having a surface with capture oligonucleotides attached thereto; and

a plurality of polynucleotide molecules attached to the surface, wherein each polynucleotide molecule comprises a concatemer of multiple copies of a target sequence and an adaptor oligonucleotide such that the polynucleotide molecule is attached to the surface by one or more complexes formed between capture oligonucleotides and adaptor oligonucleotides, the polynucleotide molecules being randomly disposed on the surface at a density such that at least a majority of the polynucleotide molecules have a nearest neighbor distance of at least fifty nm.

40. The array of claim 39 wherein said surface is a planar surface having an array of discrete spaced apart regions, wherein each discrete spaced apart region has an area less than 1 μ

m² and contains said capture oligonucleotides attached thereto and wherein substantially all such regions have at most one of said polynucleotide molecules attached.

41. The array of claim 40 wherein said discrete spaced apart regions are wells in said support, the wells each having an opening with an area equal to or less than that of said discrete spaced apart regions.

42. The array of claim 40 wherein said discrete spaced apart regions form a regular array with a nearest neighbor distance in the range of from 0.1 to 20 μ

m.

43. The array of claim 42 wherein said polynucleotide molecules on said discrete spaced apart regions have a nearest neighbor distance such that they are optically resolvable.

44. The array of claim 42 wherein each of said discrete spaced apart regions is surrounded by an inter-regional space that is substantially free of said polynucleotide molecules.

45. The array of claim 42 wherein said polynucleotide molecules are randomly distributed on said discrete spaced apart regions and wherein said nearest neighbor distance is in the range of from 0.3 to 3 μ

m.

46. The array of claim 45 wherein substantially every said discrete spaced apart region has a polynucleotide molecule attached.

47. The array of claim 45 wherein said concatemer comprises alternating copies of said target sequence and said adaptor oligonucleotide.

48. The array of claim 47 wherein each of said concatemers contains at least 10 copies of its respective target sequence.

49. The array of claim 47 wherein said target sequences each have a length in the range of from 50 to 500 nucleotides and wherein said adaptor oligonucleotide has a length in the range of from 6 to 60 nucleotides.

50. The array of claim 45 wherein all of said capture oligonucleotides have the same nucleotide sequence.

51. The array of claim 45 wherein each of said concatemers comprises at least one decoder oligonucleotide for identifying its respective target sequence.

52. The array of claim 45 wherein said discrete spaced apart regions are disposed on said planar surface in a rectilinear pattern wherein each discrete spaced apart region has an area enclosed by a circle having a diameter of from 0.1 to 10 μ

m.

53. The array of claim 45 wherein said one or more complexes between said capture oligonucleotides and said adaptor oligonucleotides are duplexes.

54. The array of claim 40 wherein said discrete spaced apart regions form a plurality of regular subarrays such that said discrete spaced apart regions within the same subarray have capture oligonucleotides having the same nucleotide sequence and said discrete space apart regions within different subarrays have capture oligonucleotides having different nucleotide sequences, and wherein within any subarray of the plurality each of said discrete spaced apart regions has a nearest neighbor distance in the range of from 1 to 20 μ

m.

55. The array of claim 54 wherein said regular subarrays are each rectilinear arrays and wherein said nearest neighbor distance between said discrete spaced apart regions is the same for each.

56. The array of claim 39 wherein said polynucleotide molecules are randomly disposed on said planar surface such that at least a majority of said polynucleotide molecules are optically resolvable.

57. The array of claim 56 wherein at least seventy percent of said polynucleotide molecules are optically resolvable.

58. A method of making an array of polynucleotide molecules, the method comprising the steps of:

generating a plurality of polynucleotide molecules each comprising a concatemer of a DNA fragment from a source DNA and an adaptor oligonucleotide;

disposing the plurality of polynucleotide molecules onto a support having a surface with capture oligonucleotides attached thereto so that the polynucleotide molecules are fixed to the surface by one or more complexes formed between capture oligonucleotides and adaptor oligonucleotides and so that the polynucleotide molecules are randomly distributed on the surface at a density such that a majority of the polynucleotide molecules have a nearest neighbor distance of at least fifty nm, thereby forming the array of polynucleotide molecules.

59. The method of claim 58 wherein said surface is a planar surface having an array of discrete spaced apart regions, wherein each discrete spaced apart region has an area of less than 1 μ

m² and contains said capture oligonucleotides attached thereto and wherein substantially all such regions have at most one of said polynucleotide molecules attached.

60. The method of claim 59 further including the step of amplifying said polynucleotide molecules after disposing them on said support.

61. The method of claim 60 wherein said step of amplifying includes cleaving said concatemers to form second fragments, circularizing the second fragments, and amplifying the second fragments by a RCR reaction within said discrete spaced apart region of each of said concatemers.

62. The array of claim 61 wherein said discrete spaced apart regions are wells in said support, the wells each having an opening with an area equal to or less than that of said discrete spaced apart regions.

63. The method of claim 59 wherein said discrete spaced apart regions form a regular array with a nearest neighbor distance in the range of from 0.1 to 20 μ

m.

64. The method of claim 63 wherein said polynucleotide molecules are randomly distributed on said discrete spaced apart regions and wherein said nearest neighbor distance is in the range of from 0.3 to 3 μ

m.

65. The method of claim 64 wherein said step of generating further includes the steps of fragmenting said source DNA to produce said DNA fragments, circularizing said DNA fragments, and forming concatemers of said DNA fragments by rolling circle replication.

66. The method of claim 65 wherein said polynucleotide molecules on said discrete spaced apart regions have a nearest neighbor distance such that they are optically resolvable.

67. A method of making an array of polynucleotide molecules, the method comprising the steps of:

generating a plurality of polynucleotide molecules each comprising a concatemer of a DNA fragment from a source nucleic acid;

disposing the plurality of polynucleotide molecules onto a support having a surface having reactive functionalities attached thereto so that the polynucleotide molecules are fixed to the surface by one or more linkages formed between the reactive functionalities and complementary functionalities on the polynucleotide molecules and so that the polynucleotide molecules are randomly disposed on the surface at a density such that at least a majority of the polynucleotide molecules have a nearest neighbor distance of at least fifty nm, thereby forming the array of polynucleotide molecules.

68. The method of 67 wherein said surface is a planar surface having an array of discrete spaced apart regions, wherein each discrete spaced apart region has an area of less than 1 μ

m² and contains said reactive functionalities attached thereto and wherein substantially all such regions have at most one of said polynucleotide molecules attached.

69. The method of claim 68 wherein said step of generating further includes the steps of fragmenting said source DNA to produce said DNA fragments, circularizing said DNA fragments, and forming concatemers of said DNA fragments by rolling circle replication.

70. A method of determining a nucleotide sequence of a target polynucleotide, the method comprising the steps of:

(a) generating a plurality of target concatemers from the target polynucleotide, each target concatemer comprising multiple copies of a fragment of the target polynucleotide and the plurality of target concatemers including a number of fragments that substantially covers the target polynucleotide;

(b) forming a random array of target concatemers fixed to a surface at a density such that at least a majority of the target concatemers are optically resolvable;

(c) hybridizing one or more probes from a first set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on target concatemers;

(d) hybridizing one or more probes from a second set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on target concatemers;

(e) ligating probes from the first and second sets hybridized to a target concatemer at contiguous sites;

(f) identifying the sequences of the ligated first and second probes; and

(g) repeating steps (c) through (f) until the sequence of the target polynucleotide can be determined from the identities of the sequences of the ligated probes.

71. The method of claim 70 wherein said surface is a planar surface having an array of discrete spaced apart regions, wherein each discrete spaced apart region has an area of less than 1 μ

m² and wherein substantially all such regions have at most one of said target concatemers attached.

72. A method of determining a nucleotide sequence of a target polynucleotide, the method comprising the steps of:

(a) providing a random array of target concatemers fixed to a planar surface having an array of optically resolvable discrete spaced apart regions, wherein each discrete spaced apart region has an area of less than 1 μ

m² and wherein substantially all such regions have at most one of said target concatemers attached, each target concatemer comprising multiple copies of a fragment of the target polynucleotide and a number of different target concatemers being present such that their respective fragments substantially cover the target polynucleotide;

(b) hybridizing one or more probes from a first set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on target concatemers;

(c) hybridizing one or more probes from a second set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on target concatemers;

(d) ligating probes from the first and second sets hybridized to a target concatemer at contiguous sites;

(e) identifying the sequences of the ligated first and second probes; and

(f) repeating steps (b) through (e) until the sequence of the target polynucleotide can be determined from the identities of the sequences of the ligated probes.

73. A method of determining a nucleotide sequence of a target polynucleotide, the method comprising the steps of:

(a) generating a plurality of target concatemers from the target polynucleotide, each target concatemer comprising multiple copies of a fragment of the target polynucleotide and the plurality of target concatemers including a number of fragments that substantially covers the target polynucleotide;

(b) forming a random array of target concatemers fixed to a surface at a density such that at least a majority of the target concatemers are optically resolvable;

(c) identifying a sequence of at least a portion of each fragment in each target concatemer; and

(d) reconstructing the nucleotide sequence of the target polynucleotide from the identities of the sequences of the portions of fragments of the concatemers.