SELF-ASSEMBLED SINGLE MOLECULE ARRAYS AND USES THEREOF

• US 20110281738A1
• Filed: 05/02/2011
• Published: 11/17/2011
• Est. Priority Date: 10/07/2005
• Status: Active Grant

• Current Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Original Assignee: Callida Genomics, Inc. (SBH Genomics, Inc.)
• Inventors: Drmanac, Radoje, Yeung, George, Callow, Matthew J., Hauser, Brian K.
• Granted Patent: US 8,609,335 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• 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 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2525/313 Branched oligonucleotides
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides

Abstract

The present invention provides methods of making and using self-assembled arrays of single polynucleotide molecules for carrying out a variety of large-scale genetic measurements, such as gene expression analysis, gene copy number assessment, and the like. Random arrays used in the invention are “self-assembled” in the sense that they are formed by deposition of polynucleotide molecules onto a surface where they become fixed at random locations. The polynucleotide molecules fixed on the surface are then identified by direct sequence determination of component nucleic acids, such as incorporated probe sequences, or by other decoding schemes. Such identification converts a random array of determinable polynucleotides, and their respective probes into an addressable array of probe sequences.

Claims

1. A method of making a probe array, the method comprising the steps of:

providing a plurality of polynucleotide molecules attached to a surface of a support, wherein each polynucleotide molecule has a random coil state and comprises a concatemer of multiple copies of a probe 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; and

identifying the probe sequence of each polynucleotide molecule on the surface to form the probe array.

2. The method of claim 1 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.

3. The method 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 polynucleotide molecule and contains said reactive functionalities attached thereto and wherein such regions have at most one of said polynucleotides attached.

4. The method of claim 3 wherein said reactive functionalities are hydrophobic functionalities.

5. The method of claim 3 wherein said discrete spaced apart region has an area of less than 1 μ

m².

6. The method of claim 5 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.

7. The method of claim 6 wherein substantially every said discrete spaced apart region has a polynucleotide attached.

8. The method of claim 6 wherein said concatemer comprises alternating copies of said probe sequence and an adaptor oligonucleotide.

9. The method of claim 8 wherein each of said concatemers contains at least 10 copies of its respective probe sequence.

10. The method of claim 8 wherein said probe sequences each have a length in the range of from 10 to 500 nucleotides and wherein said adaptor oligonucleotide has a length in the range of from 6 to 60 nucleotides.

11. The method of claim 8 wherein said probe sequences comprise mate-paired ends of restriction fragments.

12. The method of claim 6 wherein each of said concatemers comprises at least one decoder oligonucleotide for identifying its respective probe sequence.

13. The method of claim 6 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.

14. The method of claim 3 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.

15. The method of claim 14 wherein each of said discrete spaced apart regions is surrounded by an inter-regional space that is substantially free of said polynucleotides.

16. The method of claim 15 wherein said probe sequences comprise mate-paired ends of restriction fragments.

17. The method of claim 2 wherein said polynucleotides are randomly disposed on said planar surface such that at least a majority of said polynucleotides are optically resolvable.

18. The method of claim 17 wherein at least seventy percent of said polynucleotides are optically resolvable.

19. The method of claim 17 wherein said probe sequences comprise mate-paired ends of restriction fragments.

20. A method of making a probe array, the method comprising the steps of:

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

providing a plurality of polynucleotide molecules attached to the surface, wherein each polynucleotide molecule comprises a concatemer of multiple copies of a probe 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; and

identifying the probe sequence of each polynucleotide molecule on the surface to form the probe array.

21. The method of claim 20 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.

22. The method of claim 21 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.

23. The method of claim 22 wherein said polynucleotide molecules on said discrete spaced apart regions have a nearest neighbor distance such that they are optically resolvable.

24. The method of claim 22 wherein each of said discrete spaced apart regions is surrounded by an inter-regional space that is substantially free of said polynucleotide molecules.

25. The method of claim 22 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.

26. The method of claim 25 wherein substantially every said discrete spaced apart region has a polynucleotide molecule attached.

27. The method of claim 25 wherein said concatemer comprises alternating copies of said probe sequence and said adaptor oligonucleotide.

28. The method of claim 27 wherein each of said concatemers contains at least 10 copies of its respective probe sequence.

29. The method of claim 27 wherein said target sequences each have a length in the range of from 10 to 500 nucleotides and wherein said adaptor oligonucleotide has a length in the range of from 6 to 60 nucleotides.

30. The method of claim 27 wherein said probe sequences comprise mate-paired ends of restriction fragments.

31. The method of claim 25 wherein all of said capture oligonucleotides have the same nucleotide sequence.

32. The method of claim 25 wherein each of said concatemers comprises at least one decoder oligonucleotide for identifying its respective target sequence.

33. The method of claim 25 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.

34. The method of claim 25 wherein said one or more complexes between said capture oligonucleotides and said adaptor oligonucleotides are duplexes.

35. The method of claim 20 wherein said polynucleotide molecules are randomly disposed on said planar surface such that at least a majority of said polynucleotide molecules are optically resolvable.

36. The method of claim 35 wherein at least seventy percent of said polynucleotide molecules are optically resolvable.

37. The method of claim 35 wherein said probe sequences comprise mate-paired ends of restriction fragments.

38. A method of making a probe array, the method comprising the steps of:

generating a plurality of polynucleotide molecules each comprising a concatemer of a probe sequence 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; and

identifying the probe sequence of each polynucleotide molecule on the surface to form the probe array.

39. The method of claim 38 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.

40. The method of claim 39 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.

41. The method of claim 40 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.

42. The method of claim 41 wherein said step of generating further includes the steps of fragmenting a source DNA to produce probe sequences, circularizing said probe sequences, and forming concatemers of said probe sequences by rolling circle replication.

43. A method of making a probe array, the method comprising the steps of:

generating a plurality of polynucleotide molecules each comprising a concatemer of a probe sequence 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; and

identifying the probe sequence of each polynucleotide molecule on the surface to form the probe array.

46. The method of claim 43 wherein said step of generating further includes the steps of fragmenting said source nucleic acid to produce said probe sequence, circularizing said probe sequences with an adaptor oligonucleotide, and forming concatemers of said probe sequences and adaptor oligonucleotides by rolling circle replication.

47. The method of claim 43 wherein said step of generating further includes the steps of fragmenting said source nucleic acid to produce said probe sequence, circularizing said probe sequences with an adaptor oligonucleotide, inserting one or more interspersed adaptor into each probe sequence, and forming concatemers of said probe sequences, adaptor oligonucleotides, and interspersed adaptors by rolling circle replication.

44. The method of 43 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.

45. The method of claim 44 wherein said step of generating further includes the steps of fragmenting said source nucleic acid to produce said probe sequence, circularizing said probe sequences, and forming concatemers of said probe sequences by rolling circle replication.

48. A method of measuring amounts of a plurality of different target polynucleotides in a sample, the method comprising the steps of:

providing a pool of nucleic acid fragments derived from the sample containing the plurality of different target polynucleotides;

hybridizing the pool of nucleic acid fragments to a random array of probe 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 probe concatemers attached, each probe concatemer comprising multiple copies of a fragment of a target polynucleotide of the plurality, or a complement thereof, where the fragment or complement thereof is identified by a portion of its sequence; and

quantifying the hybridization of said nucleic acid fragments to the random array, wherein such quantification is proportional to the amounts of the plurality of different polynucleotides in the sample.

49. The method of claim 48 wherein said sample is a cDNA library and wherein said plurality of different polynucleotides is at least one hundred different polynucleotides.

50. The method of claim 48 wherein said random array of probe concatemers contains a number of probe concatemers such that each of said plurality of different polynucleotides has at least a portion of sequence complementary to at least one probe concatemer with a probability of at least 95 percent.

51. The method of claim 50 wherein said number of probe concatemers is at least 10⁶.

52. The method of claim 51 wherein said number of probe concatemers is at least 10⁷.

53. The method of claim 52 wherein said number of probe concatemers is at least 10⁸.

54. A method of measuring amounts of a plurality of different target polynucleotides in a sample, the method comprising the steps of:

providing a pool of nucleic acid fragments derived from the sample containing the plurality of different target polynucleotides;

hybridizing the pool of nucleic acid fragments to a random array of probe concatemers fixed to a surface at a density such that at least a majority of the probe concatemers are optically resolvable, each probe concatemer comprising multiple copies of a fragment of a target polynucleotide of the plurality or a complement thereof, where the fragment or complement thereof is identified by a portion of its sequence; and

quantifying the hybridization of said nucleic acid fragments to the random array, wherein such quantification is proportional to the amounts of the plurality of different polynucleotides in the sample.

55. The method of claim 54 wherein said random array of probe concatemers contains a number of probe concatemers such that each of said plurality of different polynucleotides has at least a portion of sequence complementary to at least one probe concatemer with a probability of at least 95 percent.

56. The method of claim 55 wherein said number of probe concatemers is at least 10⁶.

57. The method of claim 56 wherein said number of probe concatemers is at least 10⁷.

58. The method of claim 57 wherein said number of probe concatemers is at least 10⁸.