Method of sequencing a nucleic acid
First Claim
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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, wherein said solid support includes at least one optical fiber;
providing a plurality of single-stranded 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 an extended anchor primer covalently linked to a nucleic acid comprising multiple copies of a sequence complementary to the circular nucleic acid template;
annealing an effective amount of a sequencing primer to one or more copies of said covalently linked complementary nucleic acid;
extending the sequencing primer with a polymerase and a predetermined nucleotide triphosphate to yield a sequencing product and, if the predetermined nucleotide triphosphate is incorporated onto the 3′
end of said sequencing primer, a sequencing reaction byproduct; and
identifying the sequencing reaction byproduct, thereby determining the sequence of the nucleic acid.
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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.
859 Citations
32 Claims
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1. A method for sequencing a nucleic acid, the method comprising:
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providing one or more or more nucleic acid anchor primers linked to a solid support, wherein said solid support includes at least one optical fiber;
providing a plurality of single-stranded 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 an extended anchor primer covalently linked to a nucleic acid comprising multiple copies of a sequence complementary to the circular nucleic acid template;
annealing an effective amount of a sequencing primer to one or more copies of said covalently linked complementary nucleic acid;
extending the sequencing primer with a polymerase and a predetermined nucleotide triphosphate to yield a sequencing product and, if the predetermined nucleotide triphosphate is incorporated onto the 3′
end of said sequencing primer, a sequencing reaction byproduct; and
identifying the sequencing reaction byproduct, thereby determining the sequence of the nucleic acid. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)
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.
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13. The method of claim 1, wherein the sequencing byproduct is pyrophosphate.
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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.
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15. The method of claim 14, wherein the ATP is detected with luciferase.
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16. The method of claim 13, further comprising apyrase.
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17. The method of claim 13, further comprising washing the sequencing product with a wash buffer.
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18. The method of claim 17, wherein the wash buffer includes apyrase.
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19. The method of claim 1, wherein the anchor primer sequence includes a biotin group.
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20. The method of claim 19, wherein the biotin group on the anchor primer is linked to an avidin group on the solid support.
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21. The method of claim 1, wherein the anchor primer is conjugated to a biotin-BSA moiety.
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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.
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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.
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24. The method of claim 1, wherein the sequencing primer is extended in the presence of a dATP analog.
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25. 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.
- m to approximately 200 μ
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26. The method of claim 25, wherein the solid substrate includes two or more anchoring primers separated by approximately 50 μ
- m to approximately 150 μ
m.
- m to approximately 150 μ
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27. The method of claim 25, wherein the solid substrate includes two or more anchoring primers separated by approximately 100 μ
- m to approximately 150 μ
m.
- m to approximately 150 μ
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28. The method of claim 1, wherein the solid support comprises of a plurality of anchor pads that are covalently linked to the solid support.
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29. The method of claim 28, wherein the surface area of each anchor pad is approximately 10 μ
- m2.
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30. The method of claim 28, wherein and each pad is separated from one another by a distance ranging from approximately 50 μ
- m to approximately 150 μ
m.
- m to approximately 150 μ
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31. The method of claim 15, wherein the sulfurylase and luciferase are present at sufficient concentrations to detect ATP with first-order kinetics.
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32. The method of claim 31, wherein incorporation of two or more identical nucleotide triphosphates onto the 3′
- end of said sequencing primer is revealed by a corresponding fold increase in light released.
Specification