Polony fluorescent in situ sequencing beads

• US 20070087362A1
• Filed: 08/16/2006
• Published: 04/19/2007
• 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, Shendure, Jay, Porreca, Gregory
• Granted Patent: US 7,425,431 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 substantially identical nucleic acid sequences attached thereto that differs in sequence from the population of substantially identical nucleic acid sequences attached to other individual beads, 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 1, wherein the individual beads comprise two, three or four different populations of substantially identical nucleic acid sequences attached thereto.

5. The array of claim 1, wherein at least 40% of the individual beads include the population of substantially identical nucleic acid sequences attached thereto.

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

7. 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.

8. 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.

9. 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.

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.

36. The method of claim 1, wherein the substantially identical nucleic acid sequences are primers.

37. The method of claim 1, wherein the substantially identical nucleic acid sequences are amplified nucleic acid sequences.

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

a) providing a plurality of beads wherein an individual bead has a population of substantially identical nucleic acid sequences attached thereto that differs in sequence from the population of substantially identical nucleic acid sequences attached to other individual beads; and

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

14. The method of claim 13, further comprising attaching the semi-solid medium to a solid support in step b).

15. The method of claim 14, wherein the support is a microscope slide or a flow cell.

16. The method of claim 13, wherein the plurality of beads comprises two, three or four different populations of substantially identical nucleic acid sequences attached thereto.

17. The method of claim 13, wherein at least 40% of the individual beads include the population of substantially identical nucleic acid sequences attached thereto.

18. The method of claim 13, wherein the beads are immobilized as a monolayer.

19. The method of claim 13, 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.

20. The method of claim 13, wherein the semi-sold medium has top and bottom surfaces and the plurality of beads is immobilized near the top surface.

21. The method of claim 14, 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 13, 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.

23. The method of claim 13, wherein the plurality of beads comprises a plurality of clonal beads.

24. The method of claim 13, wherein the plurality of beads comprises a library.

38. The method of claim 13, wherein the substantially identical nucleic acid sequences are primers.

39. The method of claim 13, wherein the substantially identical nucleic acid sequences are amplified nucleic acid sequences.

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

a) providing a plurality of beads wherein an individual bead has a population of substantially identical nucleic acid sequences attached thereto that differs in sequence from the population of substantially identical nucleic acid sequences attached to other individual beads;

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

c) amplifying the population of substantially identical nucleic acid sequences to form a plurality of beads having an amplified population of substantially identical nucleic acid sequences attached thereto.

26. The method of claim 25, wherein the semi-solid medium includes an amplification primer.

27. The method of claim 25, wherein the semi-solid medium includes an additive that forms voids in the semi-solid medium.

28. The method of claim 27, wherein the additive is selected from the group consisting of:

cationic lipid, polyamine and polycation.

40. The method of claim 25, wherein the substantially identical nucleic acid sequences are primers.

41. The method of claim 25, wherein the substantially identical nucleic acid sequences are amplified nucleic acid sequences.

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

a) providing a plurality of beads wherein an individual bead has a population of substantially identical nucleic acid sequences attached thereto that differs in sequence from the population of substantially identical nucleic acid sequences attached to other individual beads;

b) amplifying the population of substantially identical nucleic acid sequences to form a plurality of immobilized beads having an amplified population of substantially identical nucleic acid sequences attached thereto; and

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

30. The method of claim 29, wherein the amplifying is performed by emulsion PCR.

42. The method of claim 29, wherein the substantially identical nucleic acid sequences are primers.

43. The method of claim 29, wherein the substantially identical nucleic acid sequences are amplified nucleic acid sequences.

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

a) providing a plurality of beads wherein an individual bead has a population of substantially identical nucleic acid sequences attached thereto that differs in sequence from the population of substantially identical nucleic acid sequences attached to other individual beads;

b) amplifying the population of substantially identical nucleic acid sequences to form a plurality of beads having an amplified population of substantially identical nucleic acid sequences attached thereto;

c) enriching the plurality of beads having an amplified population of substantially identical nucleic acid sequences attached thereto to form an enriched population of beads; and

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

44. The method of claim 31, wherein the substantially identical nucleic acid sequences are primers.

45. The method of claim 31, wherein the substantially identical nucleic acid sequences are amplified nucleic acid sequences.

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

a) providing a population of beads wherein an individual bead has a population of substantially identical nucleic acid sequences attached thereto that differs in sequence from the population of substantially identical nucleic acid sequences attached to other individual beads;

b) contacting the population of beads with a second nucleic acid sequence that is complementary to the first nucleic acid sequence;

c) incubating the population of beads and the second nucleic acid sequence 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.

33. The method of claim 32, wherein the second nucleic acid is immobilized on a capture bead.

34. The method of claim 32, wherein the population of hybridized beads are separated from the population of unhybridized beads by density or affinity.

46. The method of claim 32, wherein the substantially identical nucleic acid sequences are primers.

47. The method of claim 32, wherein the substantially identical nucleic acid sequences are amplified nucleic acid sequences.

35. 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 substantially identical amino acids attached thereto that differs in sequence from the population of substantially identical nucleic acid sequences attached to other individual beads.