Enzymatic light amplification

• US 20030054396A1
• Filed: 09/06/2002
• Published: 03/20/2003
• Est. Priority Date: 09/07/2001
• Status: Abandoned Application

• Current Assignee: 454 CORPORATION
• Original Assignee: 454 CORPORATION
• Inventors: Weiner, Michael P.
• Application Number:
  US10/236,871
• Publication Number:
  US 20030054396A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/682 Signal amplification
• 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 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

Reversibly labeled nucleotides and methods involving the nucleotides are disclosed. The methods included methods of determining a sequence of a nucleic acid.

Claims

1. A reversibly labeled nucleotide comprising:

(a) a nucleotide or nucleoside,

(b) at least one detectable label comprising a light generating moiety which emits light in the presence of a substrate, and

(c) a linker connecting said nucleotide or nucleoside and said detectable label wherein said linker comprise a carbon chain of between 10 carbons to 24 carbons and a cleavable bond which can be cleaved to separate (a) from (b).

2. The reversible labeled nucleotide of claim 1 wherein said nucleotide is selected from the group consisting of a nucleotide monophosphate, a nucleotide diphosphate, and a nucleotide triphosphate.

3. The reversible labeled nucleotide of claim 1 wherein said nucleotide triphosphate is selected from the group consisting of dATP, dTTP, dGTP, dCTP, ATP, UTP, GTP and CTP.

4. The reversible labeled nucleotide of claim 1 wherein said light generating moiety is connected to said linker by a specific binding pair.

5. The reversible labeled nucleotide of claim 4 wherein said binding pair is biotin/avidin, biotin/streptavidin, disulfide derivatives or functional derivatives and analogs thereof.

6. The reversible labeled nucleotide of claim 5 wherein said disulfide derivative is a disulfide derivative of biotin.

7. The reversible labeled nucleotide of claim 4 wherein said binding pair is selected from the group consisting of antigen/antibody, hapten/peptide, maltose/maltose binding protein, protein A/antibody fragment, protein G/antibody fragment, polyhistidine/nickel, glutathione S transferase/glutathione and derivatives, functional fragments, and functional analogs thereof.

8. The reversible labeled nucleotide of claim 1 wherein said light generating moiety is selected from the group consisting of green fluorescent protein, blue fluorescent protein, red fluorescent protein, beta-galactosidase, chloramphenicol acetyltransferase, beta-glucoronidase, luciferases, b-lactamase, digoxygenin, and derivatives thereof.

9. The reversible labeled nucleotide of claim 8 wherein said fluorescent dye molecule is cy3 or cy5.

10. The reversible labeled nucleotide of claim 8 wherein said derivatives are selected from the group consisting of blue EBFP, cyan ECFP, yellow-green EYFP, destabilized GFP variants, stabilized GFP variants and fusion variants

11. The reversible labeled nucleotide of claim 1 wherein said light generating moiety is alkaline phosphatase or horse radish peroxidase.

12. The reversible labeled nucleotide of claim 1 wherein said substrate is selected from the group consisting of ATP, NBR/BCIP, ascorbate, ferrocyanide, cytochrome C X-gal, Acetyl CoA, n-butytyl CoA, chloramphenicol, glucoronides, antidigoxigenin-POD, diaminobenzidine, luciferin, beta-lactam, glucuronides, H₂O₂ and a combination thereof.

13. The reversible labeled nucleotide of claim 1 wherein said at least one detectable label is detectable using chemical or enzymatic methods.

14. The reversible labeled nucleotide of claim 1 wherein said linker comprises a carbon chain of between about 18 to about 22 carbons.

15. The reversible labeled nucleotide of claim 1 wherein said linker comprises a carbon chain of about 20 carbons.

16. The reversible labeled nucleotide of claim 1 wherein said linker is connected to a sugar, a base or a phosphate moiety on said nucleotide triphosphate.

17. The reversible labeled nucleotide of claim 1 wherein said linker is connected to said nucleotide triphosphate by a cleavable bond.

18. The reversible labeled nucleotide of claim 1 wherein said detectable moiety is selected from the group consisting of fluorescent dye molecule, fluorescein and a combination thereof.

19. The reversible labeled nucleotide of claim 1 wherein said cleavable bond is a covalent or ionic bond.

20. The reversible labeled nucleotide of claim 19 wherein said cleavable bond is cleavable by exposure to a reducing agent.

21. The reversible labeled nucleotide of claim 1 wherein said reducing agent is selected form the group consisting of dithiothreitol, β

-mercaptoethanol.

22. The reversible labeled nucleotide of claim 1 wherein said cleavable bond is cleavable by exposure to heat, cold, chemical denaturants, surfactants, hydrophobic reagents, and suicide inhibitors.

23. The reversible labeled nucleotide of claim 1 wherein said detectable label can be inactivated by exposure to reducing agents, heat, cold, chemical denaturants, surfactants, hydrophobic reagents, and suicide inhibitors.

24. A method of determining the incorporation of a nucleotide into an elongating chain of a nucleic acid by:

(a) contacting a nucleic acid with a first species of a reversibly labeled nucleotide triphosphate according to claim 1;

(b) detecting incorporation of said first species of nucleotide triphosphate to said elongating chain of nucleic acid by detecting light emitted by said light generating moiety in the presence of a detection substrate.

25. The method of claim 24, further comprising the steps of:

(c) inactivating or detaching said label;

(d) repeating steps (a), (b) and (c) using a second species of nucleotide triphosphate of claim 1 wherein said first species and said second species are different.

26. The method of claim 25 further comprising the step of determining the sequence of the elongating nucleic acid by recording the order of nucleotide triphosphate used in step (a) and the results of step (b).

27. The method of claim 24 wherein said light generating moiety is selected from the group consisting of alkaline phosphatase, horse radish peroxidase, digoxygenin, fluorescent dye molecule, and fluorescein.

28. The method of claim 24 wherein said light generating moiety is selected from the group consisting of alkaline phosphatase, horse radish peroxidase, green fluorescent protein, blue fluorescent protein, red fluorescent protein, beta-galactosidase, chloramphenicol acetyltransferase, beta-glucoronidase, luciferases, b-lactamase and derivatives thereof.

29. The method of claim 24 wherein said detection substrate is selected from the group consisting of ATP, NBR/BCIP, ascorbate, ferrocyanide, cytochrome C X-gal, Acetyl CoA, n-butytyl CoA, chloramphenicol, glucoronides, antidigoxigenin-POD, diaminobenzidine, luciferin, beta-lactam, glucuronides, H₂O₂ and a combination thereof.

30. The method of claim 24 wherein said elongating nucleic acid is elongating along a template nucleic acid and wherein the sequence of the template nucleic acid is determined.

31. The method of claim 24 wherein said chain elongation reaction is a transcription reaction, a replication reaction, a reverse transcription reaction.

32. The method of claim 24 wherein said light emitted is nonstoichlometrc.

33. The method of claim 24 wherein said light emitted is greater than 1000 photons per nucleotide triphosphate incorporated.

34. The method of claim 24 wherein said light emitted is greater than 100 photons per nucleotide triphosphate incorporated.

35. The method of claim 24 wherein said light emitted is greater than 10 photons per nucleotide triphosphate incorporated.

36. A reversibly labeled nucleotide comprising:

(a) a nucleotide or nucleoside,

(b) at least one conjugatable moiety that comprises one part of a binding pair, and

(c) a linker connecting said nucleotide or nucleoside and said detectable label wherein said linker comprise a carbon chain of between 10 carbons to 24 carbons and a cleavable bond which can be cleaved to separate (a) from (b).

37. The reversible labeled nucleotide of claim 36 wherein said one part of a binding pair is selected from a group of binding pairs consisting of antigen/antibody, hapten/peptide, maltose/maltose binding protein, protein A/antibody fragment, protein G/antibody fragment, polyhistidine/nickel, glutathione S transferase/glutathione and derivatives, functional fragments, and functional analogs thereof.

38. The nucleotide of claim 36 or 37 further comprising a detectable label connected to a complementary part of said one part of a binding pair.

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

(a) providing one or more nucleic acid anchor primers;

(b) providing a plurality of single-stranded nucleic acid templates disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m;

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

(d) combining the primed anchor primer-template complex with a polymerase to form an extended anchor primer covalently linked to multiple copies of a nucleic acid complementary to the nucleic acid template;

(e) annealing an effective amount of a sequencing primer to one or more copies of said covalently linked complementary nucleic acid;

(f) extending the sequencing primer with a polymerase and a predetermined reversibly labeled nucleotide triphosphate according to claim 36 or 38 to yield a sequencing product; and

(g) detecting the amount of incorporation of said reversibly labeled triphosphate, thereby determining the sequence of the nucleic acid.

40. The method of claim 39 wherein said detecting step comprises the steps of

(a) contacting said nucleic acid with a detectable label a detectable label connected to a complementary part of said one part of a binding pair;

(b) detecting the incorporation of said detectable label to said extended sequencing primer.

41. The method of claim 39 or 40 further comprising the step of removing said detectable label after said detecting step.

42. The method of claim 41 wherein said step of removing said detectable label comprises exposing said label to a reducing agent.

43. The method of claim 42 wherein said reducing agent is selected form the group consisting of dithiothreitol, β

-mercaptoethanol.

44. The method of claim 42 wherein said step of removing comprise exposing said label to heat, cold, chemical denaturants, surfactants, hydrophobic reagents, and suicide inhibitors.

45. The method of claim 39 or 40 further comprising the step of inactivating said detectable label.

46. The method of claim 45 wherein said step of inactivating said detectable label comprise exposing said label to heat, cold, chemical denaturants, surfactants, hydrophobic reagents, and suicide inhibitors.

47. The method of claim 39 wherein each single stranded nucleic acid is circular.

48. The method of claim 39 wherein each single stranded circular nucleic acid contains at least 100 copies of a nucleic acid sequence, each copy covalently linked end to end.

49. The method of claim 39 wherein each reaction chamber has a width in at least one dimension of between 0.3 μ

m and 100 μ

m.

50. The method of claim 39 wherein each reaction chamber has a width in at least one dimension of between 0.3 μ

m and 20 μ

m.

51. The method of claim 39 wherein each reaction chamber has a width in at least one dimension of between 0.3 μ

m and 10 μ

m.

52. The method of claim 39 wherein each reaction chamber has a width in at least one dimension of between 20 μ

m and 70 μ

m.

53. The method of claim 39 wherein the cavities number greater than 400,000.

54. The method of claim 39 wherein the cavities number between 400,000 and 20,000,000.

55. The method of claim 39 wherein the cavities number between 1,000,000 and 16,000,000.

56. The method of claim 39 wherein the center to center spacing is between 10 to 150 μ

m.

57. The method of claim 39 wherein the center to center spacing is between 50 to 100 μ

m.

58. The method of claim 39, wherein each cavity has a depth of between 10 μ

m and 100 μ

m.

59. The method of claim 39 wherein each cavity has a depth that is between 0.25 and 5 times the size of the width of the cavity.

60. The method of claim 39 wherein each cavity has a depth that is between 0.3 and 1 times the size of the width of the cavity.

61. The method of claim 39 wherein the nucleic acid sequence is further amplified to produce multiple copies of said nucleic acid sequence after being disposed in the reaction chamber.

62. The method of claim 61 wherein the nucleic acid sequence is amplified using an amplification technology selected from the group consisting of polymerase chain reaction, ligase chain reaction and isothermal DNA amplification.

63. The method of claim 39 wherein the single stranded nucleic acid is immobilized in the reaction chamber.

64. The method of claim 39 wherein the single stranded nucleic acid is immobilized on one or more mobile solid supports disposed in the reaction chamber.

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

(a) providing at least one nucleic acid anchor primer;

(b) providing a plurality of single-stranded circular nucleic acid templates in an array having at least 400,000 discrete reaction sites;

(c) annealing a first 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;

(d) combining the primed anchor primer-circular template complex with a polymerase to form an extended anchor primer covalently linked to multiple copies of a nucleic acid complementary to the circular nucleic acid template;

(e) annealing a second amount of a sequencing primer to one or more copies of the covalently linked complementary nucleic acid;

(f) extending the sequencing primer with a polymerase and a predetermined nucleotide triphosphate according to claim 36 or 38 to yield a sequencing product and, when the predetermined nucleotide triphosphate is incorporated onto the 3′

end of the sequencing primer; and

(g) identifying the detectable label, thereby determining the sequence of the nucleic acid at each reaction site that contains a nucleic acid template.

66. The method of claim 65 further comprising the step of:

(h) removing or inactivating said detectable label.

67. The method of claim 66 further comprising the step of repeating steps (f) (g) and (h) with a different labeled nucleotide triphosphate.

68. The method of claim 65, wherein the anchor primer is linked to a particle.

69. The method of claim 68, wherein the anchor primer is linked to the particle prior to formation of the extended anchor primer.

70. The method of claim 68, wherein the anchor primer is linked to the particle after formation of the extended anchor primer.

71. A method of determining the base sequence of a plurality of nucleotides on an array, the method comprising:

(a) providing a plurality of sample DNAs, each disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m,

(b) adding a nucleotide 5′

-triphosphate precursor according to claim 38 or 39, wherein said nucleotide is of one known nitrogenous base to a reaction mixture in each reaction chamber, each reaction mixture comprising a template-directed nucleotide polymerase and a single-stranded polynucleotide template hybridized to a complementary oligonucleotide primer strand at least one nucleotide residue shorter than the templates to form at least one unpaired nucleotide residue in each template at the 3′

-end of the primer strand, under reaction conditions which allow incorporation of the nucleoside 5′

-triphosphate precursor onto the 3′

-end of the primer strands, provided the nitrogenous base of the nucleoside 5′

-triphosphate precursor is complementary to the nitrogenous base of the unpaired nucleotide residue of the templates;

(c) detecting the incorporation of the reversible label to determine whether or not nucleoside 5′

-triphosphate precursor was incorporated into the primer strands indicating that the unpaired nucleotide residue of the template has a nitrogenous base composition that is complementary to that of the incorporated nucleoside 5′

-triphosphate precursor;

(d) removing or inactivating said reversible label; and

(e) sequentially repeating steps (b), (c) and (d), wherein each sequential repetition adds and, detects the incorporation of said one type of a reversibly labeled nucleotide precursor of known nitrogenous base composition; and

(f) determining the base sequence of the unpaired nucleotide residues of the template in each reaction chamber from the sequence of incorporation of said nucleoside precursors.

72. The method of claim 71 further comprising the step of removing said reversible termination before or after step (d).

73. A method for determining the nucleic acid sequence in a template nucleic acid polymer, comprising:

(a) introducing a plurality of template nucleic acid polymers into a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 5 to 200 μ

m, each reaction chamber having a polymerization environment in which the nucleic acid polymer will act as a template polymer for the synthesis of a complementary nucleic acid polymer when nucleotides are added;

(b) successively providing to the polymerization environment a series of feedstocks, each feedstock comprising a reversibly labeled nucleotide of claim 36 or 38 selected from among the nucleotides from which the complementary nucleic acid polymer will be formed, such that if the nucleotide in the feedstock is complementary to the next nucleotide in the template polymer to be sequenced said reversibly labeled nucleotide will be incorporated into the complementary polymer;

(c) detecting the incorporation of said label to determine the identify of each nucleotide in the complementary polymer and thus the sequence of the template polymer.