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1. (WO2013185137) MODIFIED BASE DETECTION WITH NANOPORE SEQUENCING
Nota: Texto obtenido mediante procedimiento automático de reconocimiento óptico de caracteres.
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What is claimed is:

1. A method for sequencing a nucleic acid template and identifying modified bases therein comprising:

providing a substrate having an upper solution above the substrate and a lower solution below the substrate, the substrate comprising a nanopore connecting the upper solution and lower solution, the nanopore sized to pass a single stranded nucleic acid;

providing a voltage across the nanopore to produce a measurable current flow through the nanopore;

controlling the rate of translation of a single stranded portion of the template nucleic acid through the pore with a processive enzyme associated with a template nucleic acid;

measuring the current through the nanopore over time as it is translated through the nanopore;

determining the sequence of a portion of the template nucleic acid as it translates through the pore using the measured current over time; and

determining the presence of modified nucleic acids in the template nucleic acid by correlating changes in the rate of transport of the nucleic acid to changes through the nanopore to the kinetics of the processive enzyme from the interaction of the modified base with the processive enzyme.

2. The method of claim 1 wherein the template nucleic acid is sequenced multiple times.

3. The method of any of the above claims wherein the processive enzyme comprises

polymerase, exonuclease, or helicase activity.

4. The method of any of the above claims wherein the processive enzyme comprises a DNA polymerase.

5. The method of claim 4 wherein the DNA polymerase has 3' to 5' exonuclease activity.

6. The method claims 1-3 wherein the processive enzyme comprises a helicase.

7. A method for sequencing a nucleic acid comprising:

providing a substrate having an upper solution above the substrate and a lower solution below the substrate, the substrate comprising a nanopore connecting the upper solution and lower solution, the nanopore sized to pass a single stranded nucleic acid;

providing a voltage across the nanopore to produce a measurable current flow through the nanopore;

controlling the rate of translation of a single stranded portion of the template nucleic acid through the pore with a processive enzyme associated with a template nucleic acid;

measuring the current through the nanopore over time as it is translated through the nanopore; and

determining the sequence of a portion of the template nucleic acid as it translates through the pore using the measured current over time;

wherein the template nucleic acid comprises hemi-genomic DNA comprising a genomic strand and a nascent strand.

8. The method of claim 7 wherein the nascent strand is translated through the pore.

9. The method of claim 7 wherein the genomic strand is translated through the pore.

10. The method of claims 7-9 wherein the genomic strand and nascent strand are attached through a hairpin loop and both strands are translated through the pore.

11. The method of claims 7-10 wherein the processive enzyme comprises polymerase,

exonuclease, or helicase activity.

12. The method of claims 7-10 wherein the processive enzyme comprises a DNA

polymerase.

13. The method of claim 12 wherein the DNA polymerase has 3' to 5' exonuclease activity.

14. The method of claims 7-10 wherein the processive enzyme comprises a helicase.

15. The method of claim 7 wherein the template nucleic acid is sequenced multiple times.

16. A nucleic acid template for nanopore sequencing comprising a strand comprising natural nucleic acid sequence and a synthetic nucleic acid sequence that is complementary to the natural nucleic acid sequence.

17. The nucleic acid template of claim 16 comprising the natural sequence hybridized to the synthetic sequence, and the sequences connected through a hairpin loop.

18. The nucleic acid template of claim 16 wherein the natural sequence and the synthetic sequence are part of a nucleic acid strand that is hybridized to a synthetic strand.

19. The nucleic acid template of claims 16-18 further comprising a second synthetic

sequence that is substantially identical with the natural sequence.

20. The nucleic acid template of claims 16-19 wherein the natural sequence is 5' of the synthetic sequence.

21. The nucleic acid template of claims 16-19 wherein the natural sequence is 3' of the synthetic sequence.

22. The nucleic acid template of claims 16-21 wherein the nucleic acid comprises DNA and the natural sequence comprises a genomic sequence.

23. A method for sequencing a nucleic acid comprising:

providing a substrate having an upper solution above the substrate and a lower solution below the substrate, the substrate comprising a nanopore connecting the upper solution and lower solution, the nanopore sized to pass a single stranded nucleic acid;

providing a voltage across the nanopore to produce a measurable current flow through the nanopore;

controlling the rate of translation of a single stranded portion of a template nucleic acid through the pore with a processive enzyme associated with the template nucleic acid;

measuring the current through the nanopore over time as it is translated through the nanopore; and

determining the sequence of a portion of the template nucleic acid as it translates through the pore using the measured current over time;

wherein the template nucleic acid comprises a strand comprising natural nucleic acid sequence and a synthetic nucleic acid sequence that is complementary to the natural nucleic acid sequence, whereby both the natural nucleic acid sequence and the synthetic nucleic acid sequences are translated through the pore.

24. The method of claim 23 further comprising using the sequence information from the synthetic sequence to identify a modified base present in the natural nucleic acid sequence.

25. The method of claim 24 wherein the template nucleic acid further comprises a second synthetic sequence that is substantially identical with the natural sequence.

26. The method of claims 23-25 wherein the processive enzyme comprises polymerase, exonuclease, or helicase activity.

27. The method of claims 23-25 wherein the processive enzyme comprises a DNA

polymerase.

28. The method of claims 23-25 wherein the processive enzyme comprises a helicase.

29. The method of claims 23-28 wherein the template nucleic acid is sequenced multiple times.

30. A method for nanopore sequencing with reduced error comprising;

providing a substrate having an upper solution above the substrate and a lower solution below the substrate, the substrate comprising a plurality of nanopores connecting the upper solution and lower solution, the nanopores sized to pass single stranded nucleic acids;

providing a voltage across the nanopores to produce a measurable current flow through the nanopores;

measuring the current through the nanopores over time as the nucleic acid templates are translated through the nanopore;

measuring the sequence of a portion of a plurality template nucleic acids as they translate through the pore using the measured current over time; wherein some of the plurality of template nucleic acids comprise the same sequence, and

wherein the sequence of some of the plurality of nucleic acids is measured under one set of reaction conditions, and the sequence of some of the plurality of nucleic acids is measured under a second set of reaction conditions, where the first and second reaction conditions each provide different error profiles, and

determining a sequence by combining the measured sequences under the first and second reaction conditions to obtain a reduced error rate than for a sequence determined under one reaction condition.

31. The method of claim 30 further comprising controlling the rate of translation of a single stranded portion of the template nucleic acid through the pore with a processive enzyme associated with a template nucleic acid.

32. The method of claims 30-31 wherein the two reaction conditions comprise two different types of nanopores.

33. The method of claim 31 wherein the two reaction conditions comprise two different types of processive enzymes.

34. The method of claims 30-33 wherein the combined sequences represent sequences on a single molecule.

35. The method of claims 30-33 wherein the combined sequences represent sequences on different molecules.

36. The method of claims 30-35 wherein the change in reaction condition comprises a

change in the temperature, pH, or in the level of divalent cation or a combination of these.