Method of sequencing a nucleic acid

• US 20020012933A1
• Filed: 04/04/2001
• Published: 01/31/2002
• Est. Priority Date: 09/16/1999
• Status: Abandoned Application

• Current Assignee: CuraGen Corporation (Celldex Therapeutics Incorporated)
• Original Assignee: CuraGen Corporation (Celldex Therapeutics Incorporated)
• Inventors: Rothberg, Jonathan M., Bader, Joel S.
• Application Number:
  US09/826,141
• Publication Number:
  US 20020012933A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• 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 2531/125 Rolling circle
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)

Abstract

Disclosed herein are methods and apparatuses for sequencing a nucleic acid. The method includes annealing a population of circular nucleic acid molecules to a plurality of anchor primers linked to a solid support, and amplifying those members of the population of circular nucleic acid molecules which anneal to the target nucleic acid, and then sequencing the amplified molecules by detecting the presence of a sequence byproduct.

Claims

1. A method for sequencing a nucleic acid, the method comprising:

providing one or more or more nucleic acid anchor primers linked to a solid support;

providing a plurality of circular nucleic acid templates;

annealing an effective amount of the nucleic acid anchor primer to at least one of the single-stranded circular templates to yield a primed anchor primer-circular template complex;

combining the primed anchor primer-circular template complex with a polymerase to generate multiple copies of the circular nucleic acid template;

annealing an effective amount of a sequencing primer to the circular nucleic acid template to yield a primed sequencing primer-circular nucleic acid template complex;

extending the sequencing primer with a polymerase and a predetermined nucleotide triphosphate to yield a sequencing product and a sequencing reaction byproduct; and

identifying the sequencing reaction byproduct, thereby determining the sequence of the nucleic acid.

2. The method of claim 1, wherein the circular nucleic acid template is single-stranded DNA.

3. The method of claim 1, wherein the circular nucleic acid template is an open circle nucleic acid.

4. The method of claim 1, wherein the circular nucleic acid template is a closed circle nucleic acid.

5. The method of claim 1, wherein the circular nucleic acid template is genomic DNA.

6. The method of claim 1, wherein the circular nucleic acid template is cDNA.

7. The method of claim 1, wherein the circular nucleic acid is 10-200 nucleotides in length.

8. The method of claim 1, wherein the circular nucleic acid is 10-100 nucleotides in length.

9. The method of claim 1, wherein the circular nucleic acid is 10-50 nucleotides in length.

10. The method of claim 1, wherein the multiple copies are generated by a polymerase chain reaction.

11. The method of claim 1, wherein the primed circular template is extended by rolling circle amplification to yield a single-stranded concatamer of the annealed circular nucleic acid template.

12. The method of claim 11, further comprising:

annealing a reverse primer to the single-stranded concatamer to yield a primed concatamer template, and

combining the primed concatamer template with a polymerase enzyme to generate multiple copies of the concatamer template.

13. The method of claim 1, wherein the sequencing byproduct is pyrophosphate.

14. The method of claim 13, wherein the pyrophosphate is detected by contacting the sequencing byproduct with ATP sulfurylase under conditions sufficient to form ATP.

15. The method of claim 14, wherein the ATP is detected with luciferase.

16. The method of claim 13, further comprising apyrase.

17. The method of claim 13, further comprising washing the sequencing product with a wash buffer.

18. The method of claim 17, wherein the wash buffer includes apyrase.

19. The method of claim 1, wherein the anchor primer sequence includes a biotin group.

20. The method of claim 19, wherein the biotin group on the anchor primer is linked to an avidin group on the solid support.

21. The method of claim 1, wherein the anchor primer is conjugated to a biotin-BSA moiety.

22. The method of claim 21, wherein the biotin-BSA moiety on the anchor primer is linked to an avidin-biotin group on the solid support.

23. The method of claim 21, wherein the biotin-BSA moiety on the anchor primer is linked to a BSA group on the solid support in the presence of silane.

24. The method of claim 1, wherein the solid support includes at least one optical fiber.

25. The method of claim 1, wherein the sequencing primer is extended in the presence of a dATP analog.

26. The method of claim 25, wherein the dATP analog is adenosine 5′

-phosphosulfate (APS).

27. The method of claim 1, wherein the solid substrate includes two or more anchoring primers separated by approximately 10 μ

m to approximately 200 μ

m.

28. The method of claim 27, wherein the solid substrate includes two or more anchoring primers separated by approximately 50 μ

m to approximately 150 μ

m.

29. The method of claim 27, wherein the solid substrate includes two or more anchoring primers separated by approximately 100 μ

m to approximately 150 μ

m.

30. The method of claim 1, wherein the solid support matrix comprises of a plurality of anchor pads that are covalently linked to the solid support.

31. The method of claim 30, wherein the surface area of each anchor pad is approximately 10 μ

m².

32. The method of claim 30, wherein and each pad is separated from one another by a distance ranging from approximately 50 μ

m to approximately 150 μ

m.

43. The substrate as recited in claim 1 wherein said substrate comprises 10⁵ or more different groups of oligonucleotides with known sequences in discrete known regions.

44. The substrate as recited in claim 1 wherein the groups of oligonucleotides are attached to the surface by a linker.

33. A substrate for analyzing a nucleic acid, the substrate comprising:

a cavitated fiber optic surface; and

a nucleic acid sequence linked to the fiber optic surface.

34. The substrate of claim 33, wherein the substrate comprises a plurality of fiber optic surfaces.

35. The substrate of claim 33, wherein the nucleic acid sequence is an anchor primer.

36. The substrate of claim 33, wherein the fiber optic surface includes two or more anchoring primers separated by approximately 10 μ

m to approximately 200 μ

m.

37. The substrate of claim 33, wherein the fiber optic surface includes two or more anchoring primers separated by approximately 100 μ

m to approximately 150 μ

m.

38. The substrate of claim 33, wherein the fiber optic surface includes two or more anchoring primers separated by approximately 150 μ

m.

39. The substrate of claim 33, wherein the fiber optic surface includes two or more anchor pads separated by approximately 100 μ

m to approximately 150 μ

m.

40. The substrate of claim 39, wherein the surface area of each pad is approximately 10 μ

m².

41. A substrate with a cavitated surface comprising 10³ or more groups of oligonucleotides covalently attached to the surface in discrete known regions, the 10³ or more groups of oligonucleotides occupying a total area of less than 1 cm² on said substrate, said groups of oligonucleotides having different nucleotide sequences.

42. The substrate as recited in claim 41 wherein said substrate comprises 10⁴ or more different groups of sequences in discrete known regions.

45. An array of more than 1,000 different groups of oligonucleotide molecules with known sequences covalently coupled to a surface of a cavitated substrate, said groups of oligonucleotide molecules each in discrete known regions and differing from other groups of oligonucleotide molecules in monomer sequence, each of said discrete known regions being an area of less than about 0.01 cm² and each discrete known region comprising oligonucleotides of known sequence, said different groups occupying a total area of less than 1 cm².

46. The array as recited in claim 45 wherein said area is less than 10,000 microns².

47. The array as recited in claim 46 made by the process of:

exposing a first region of said substrate to light to remove photoremovable group from nucleic acids in said first region, and not exposing a second region of said surface to light;

covalently coupling a first nucleotide to said nucleic acids on said part of said substrate exposed to light, said first nucleotide covalently coupled to said photoremovable group;

exposing a part of said first region of said substrate to light, and not exposing another part of said first region of said substrate to light to remove said photoremovable groups;

covalently coupling a second nucleotide to said part of said first region exposed to light; and

repeating said steps of exposing said substrate to light and covalently coupling nucleotides until said more than 500 different groups of nucleotides are formed on said surface.

48. The array as recited in claim 46 comprising more than 10,000 groups of oligonucleotides of known sequences.

49. An apparatus for analyzing a nucleic acid sequence, the apparatus comprising:

a perfusion chamber, wherein the chamber includes a nucleic acid substrate;

a conduit in communication with the perfusion chamber;

an imaging system in communication with the perfusion chamber; and

a data collection system in communication with the imaging system.

50. The apparatus of claim 49, wherein the substrate is a planar substrate.

51. The apparatus of claim 49, wherein the imaging system is a fiber optic system.

52. The apparatus of claim 49, wherein the substrate comprises

a cavitated fiber optic surface in communication with said imaging system;

and

a nucleic acid sequence linked to the fiber optic surface.

53. The apparatus of claim 49, wherein the substrate comprises a plurality of fiber optic surfaces, said fiber optic surfaces being in communication with said imaging system.

54. The apparatus of claim 49, wherein the fiber optic surface includes two or more anchoring primers separated by approximately 100 μ

m to approximately 150 μ

m.

55. The apparatus of claim 49, wherein the fiber optic surface includes two or more anchoring primers separated by approximately 150 μ

m.

56. The apparatus of claim 49, wherein the fiber optic surface includes two or more anchor pads separated by approximately 100 μ

m to approximately 150 μ

m.

57. The apparatus of claim 49, wherein the surface area of each pad is approximately 5 μ

m² to approximately 20 μ

m².

58. The apparatus of claim 49, wherein the surface area of each pad is approximately 10 μ

m².

59. An apparatus for processing a plurality of analyses, the apparatus comprising:

a flow chamber having disposed therein a substrate comprising a plurality of cavitated surfaces, said cavitated surfaces having disposed thereon nucleic acid molecules;

fluid means for delivering processing reagents from one or more reservoirs to the flow chamber so that the analytes anchored to the plurality of microparticles are exposed to the reagents; and

detection means for detecting a sequence of optical signals from each microparticle of the plurality, each optical signal of the sequence being indicative of an interaction between a processing reagent and the analyte anchored thereto, wherein said detection means is in communication with the cavitated surfaces.

60. The apparatus of claim 59, wherein said detection means further comprises signal tracking means for correlating said optical signals from each of said microparticles in each of said digital images to form for each said microparticle of said plurality a sequence of said optical signals.

61. The apparatus of claim 60, wherein said signal tracking means is a CCD camera.

62. The apparatus of claim 59, wherein said analyte is DNA.