Polony fluorescent in situ sequencing beads

• US 7,425,431 B2
• Filed: 08/16/2006
• Issued: 09/16/2008
• Est. Priority Date: 02/27/2004
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Zhu, Jun, Church, George M., Shendure, Jay, Porreca, Gregory J.
• Primary Examiner(s): Martinell; James
• Application Number:
  US11/505,073
• Publication Number:
  US 20070087362A1
• Time in Patent Office: 762 Days
• Field of Search: None
• US Class Current: 435/91.1
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C12Q 2565/601 being a microscope, e.g. atomic force microscopy [AFM]

Abstract

Miniaturized, high-density, bead-based arrays are provided. Methods of producing and using clonal beads and producing and using miniaturized, high density, bead-based arrays are also provided.

Claims

1. An array comprising a plurality of beads, wherein an individual bead has a population of nucleic acid molecules having at least 50% sequence identity to each other attached thereto, which molecules differ in sequence from those of a population of nucleic acid molecules having at least 50% sequence identity to each other attached to another individual bead included in such plurality, and wherein the plurality of beads is immobilized in a semi-solid medium to form an array.

2. The array of claim 1, wherein the semi-solid medium is attached to a solid support.

3. The array of claim 2, wherein the support is a microscope slide or a flow cell.

4. The array of claim 2, wherein the semi-sold medium has top and bottom surfaces and the solid support is attached to the bottom surface.

5. The array of claim 1, wherein the individual beads comprise two, three or four different populations of nucleic acid molecules attached thereto, which molecules have at least 50% sequence identity to each other.

6. The array of claim 1, wherein at least 40% of the individual beads have populations of nucleic acid molecules attached thereto, wherein the nucleic acid molecules within each such population have at least 50% sequence identity to each other.

7. The array of claim 1, wherein the beads are immobilized as a monolayer.

8. The array of claim 1, wherein the semi-solid medium has x, y and z axes, and the plurality of beads is randomly arranged relative to the x and y axes.

9. The array of claim 1, wherein the semi-sold medium has top and bottom surfaces and the plurality of beads is immobilized near the top surface.

10. The array of claim 1, wherein the semi-solid medium is selected from the group consisting of:

polyacrylamide, cellulose, polyamide, cross-linked agarose, cross-linked dextran and cross-linked polyethylene glycol.

11. The array of claim 1, wherein the plurality of beads comprises a plurality of clonal beads.

12. The array of claim 1, wherein the plurality of beads comprises a library.

13. The array of claim 1, wherein the nucleic acid molecules having at least 50% sequence identity to each other are primers.

14. The array of claim 1, wherein the nucleic acid molecules having at least 50% sequence identity to each other are amplified nucleic acid molecules.

15. The array of claim 1, wherein each of two, three or four individual beads has a population of nucleic acid molecules attached thereto having at least 50% sequence identity to each other.

16. The array of claim 15, wherein each population of nucleic acid molecules attached to each of the two, three or four individual beads differs in sequence from one another.

17. The array of claim 15, wherein each population of nucleic acid molecules attached to each of the two, three or four individual beads is identical in sequence to one another.

18. A method of producing an array comprising the steps of:

a) providing a plurality of beads wherein an individual bead has a population of nucleic acid molecules having at least 50% sequence identity to each other attached thereto, which molecules differ in sequence from those of a population of nucleic acid molecules having at least 50% sequence identity to each other attached to another individual bead included in such plurality; and

b) immobilizing the beads in a semi-solid medium to form an array.

19. The method of claim 18, further comprising attaching the semi-solid medium to a solid support in step b).

20. The method of claim 19, wherein the support is a microscope slide or a flow cell.

21. The method of claim 19, wherein the semi-sold medium has top and bottom surfaces and the solid support is attached to the bottom surface.

22. The method of claim 18, wherein the plurality of beads comprises two, three or four different populations of nucleic acid molecules attached thereto, which molecules have at least 50% sequence identity to each other.

23. The method of claim 18, wherein at least 40% of the individual beads have populations of nucleic acid molecules attached thereto, wherein the nucleic acid molecules within each population have at least 50% sequence identity to each other.

24. The method of claim 18, wherein the beads are immobilized as a monolayer.

25. The method of claim 18, wherein the semi-solid medium has x, y and z axes, and the plurality of beads is randomly arranged relative to the x and y axes.

26. The method of claim 18, wherein the semi-sold medium has top and bottom surfaces and the plurality of beads is immobilized near the top surface.

27. The method of claim 18, wherein the semi-solid medium is selected from the group consisting of:

polyacrylamide, cellulose, polyamide, cross-linked agarose, cross-linked dextran and cross-linked polyethylene glycol.

28. The method of claim 18, wherein the plurality of beads comprises a plurality of clonal beads.

29. The method of claim 18, wherein the plurality of beads comprises a library.

30. The method of claim 18, wherein the nucleic acid molecules having at least 50% sequence identity to each other are primers.

31. The method of claim 18, wherein the nucleic acid molecules having at least 50% sequence identity to each other are amplified nucleic acid molecules.

32. The method of claim 18, wherein each of two, three or four individual beads has a population of nucleic acid molecules attached thereto having at least 50% sequence identity to each other.

33. The method of claim 32, wherein each population of nucleic acid molecules attached to each of the two, three or four individual beads differs in sequence from one another.

34. The method of claim 32, wherein each population of nucleic acid molecules attached to each of the two, three or four individual beads is identical in sequence to one another.

35. A method of producing an array comprising the steps of:

a) providing a plurality of beads wherein an individual bead has a population of nucleic acid molecules having at least 50% sequence identity to each other attached thereto, which molecules differ in sequence from those of a population of nucleic acid molecules having at least 50% sequence identity to each other attached to another individual bead included in such plurality;

b) immobilizing the beads in a semi-solid medium to form an array; and

c) amplifying the populations of nucleic acid molecules having at least 50% sequence identity to each other to form a plurality of beads having amplified populations of nucleic acid molecules having at least 50% sequence identity to each other attached thereto.

36. The method of claim 35, wherein the semi-solid medium includes an amplification primer.

37. The method of claim 35, wherein the semi-solid medium includes an additive that forms voids in the semi-solid medium.

38. The method of claim 37, wherein the additive is selected from the group consisting of:

cationic lipid, polyamine and polycation.

39. The method of claim 35, wherein the nucleic acid molecules having at least 50% sequence identity to each other are primers.

40. The method of claim 35, wherein the nucleic acid molecules having at least 50% sequence identity to each other are amplified nucleic acid molecules.

41. A method of producing an array comprising the steps of:

a) providing a plurality of beads wherein an individual bead has a population of nucleic acid molecules having at least 50% sequence identity to each other attached thereto, which molecules differ in sequence from those of a population of nucleic acid molecules having at least 50% sequence identity to each other attached to another individual bead included in such plurality;

b) amplifying the populations of nucleic acid molecules having at least 50% sequence identity to each other to form a plurality of immobilized beads having amplified populations of nucleic acid molecules having at least 50% sequence identity to each other attached thereto; and

c) immobilizing the beads in a semi-solid medium to form an array.

42. The method of claim 41, wherein the amplifying is performed by emulsion PCR.

43. The method of claim 41, wherein the nucleic acid molecules having at least 50% sequence identity to each other are primers.

44. The method of claim 41, wherein the nucleic acid molecules having at least 50% sequence identity to each other are amplified nucleic acid molecules.

45. A method of producing an array comprising the steps of:

a) providing a plurality of beads wherein an individual bead has a population of nucleic acid molecules having at least 50% sequence identity to each other attached thereto, which molecules differ in sequence from those of a population of nucleic acid molecules having at least 50% sequence identity to each other attached to another individual bead included in such plurality;

b) amplifying the populations of nucleic acid molecules having at least 50% sequence identity to each other to form a plurality of beads having amplified populations of nucleic acid molecules having at least 50% sequence identity to each other attached thereto;

c) enriching the plurality of beads having amplified populations of nucleic acid molecules having at least 50% sequence identity to each other attached thereto to form an enriched population of beads; and

d) immobilizing the beads in a semi-solid medium to form an array.

46. The method of claim 45, wherein the nucleic acid molecules having at least 50% sequence identity to each other are primers.

47. The method of claim 45, wherein the nucleic acid molecules having at least 50% sequence identity to each other are amplified nucleic acid molecules.

48. A method for enriching a population of beads having a first nucleic acid molecule attached thereto comprising the steps of:

a) providing a population of beads wherein an individual bead has a population of nucleic acid molecules having at least 50% sequence identity to each other attached thereto, which molecules differ in sequence from those of a population of nucleic acid molecules having at least 50% sequence identity to each other attached to another individual bead included in such plurality;

b) contacting the population of beads with a second nucleic acid molecule immobilized on a support, wherein the second nucleic acid molecule has a sequence that is complementary to that of the first nucleic acid molecule;

c) incubating the population of beads and the second nucleic acid molecule together such that hybridization occurs to form a population of hybridized beads and a population of unhybridized beads; and

d) separating the population of hybridized beads from the population unhybridized beads.

49. The method of claim 48, wherein the second nucleic acid is immobilized on a capture bead.

50. The method of claim 48, wherein the population of hybridized beads are separated from the population of unhybridized beads by density or affinity.

51. The method of claim 48, wherein the nucleic acid molecules having at least 50% sequence identity to each other are primers.

52. The method of claim 48, wherein the nucleic acid molecules having at least 50% sequence identity to each other are amplified nucleic acid molecules.

53. A kit containing an array comprising a plurality of beads immobilized in a semi-solid medium, wherein the plurality of beads includes an individual bead having a population of nucleic acid molecules having at least 50% sequence identity to each other attached thereto, which molecules differ in sequence from those of a population of nucleic acid sequences molecules having at least 50% sequence identity to each other attached to another individual bead included in such plurality.