Methods for sequencing individual nucleic acids under tension
First Claim
1. A method for determining incorporation of one or more nucleotides into a nucleic acid comprisingcontacting an immobilized nucleic acid with a plurality of identical nucleotides in the presence of a polymerase, anddetermining whether incorporation of one or more of the plurality of identical nucleotides occurs in a newly synthesized nucleic acid hybridized to the immobilized nucleic acid based on a change in length of the immobilized nucleic acid while it is under tension.
2 Assignments
0 Petitions
Accused Products
Abstract
The invention provides apparatuses and methods of use thereof for sequencing nucleic acids subjected to a force, and thus considered under tension. The methods may employ but are not dependent upon incorporation of extrinsically detectably labeled nucleotides.
310 Citations
78 Claims
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1. A method for determining incorporation of one or more nucleotides into a nucleic acid comprising
contacting an immobilized nucleic acid with a plurality of identical nucleotides in the presence of a polymerase, and determining whether incorporation of one or more of the plurality of identical nucleotides occurs in a newly synthesized nucleic acid hybridized to the immobilized nucleic acid based on a change in length of the immobilized nucleic acid while it is under tension.
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4-21. -21. (canceled)
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22. A method for sequencing a nucleic acid comprising
immobilizing a nucleic acid that comprises a plurality of tandemly arranged identical nucleic acids, performing a sequencing-by-synthesis reaction using the immobilized nucleic acid as a template, and detecting incorporation of nucleotides by changes in length of the nucleic acid.
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23. A method for sequencing a nucleic acid comprising
immobilizing a nucleic acid that comprises a plurality of tandemly arranged identical nucleic acids onto a support surface, performing a sequencing-by-synthesis reaction using the immobilized nucleic acid as a template, and detecting incorporation of nucleotides non-enzymatically and in the absence of fluorescence detection.
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24-27. -27. (canceled)
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28. A method for sequencing a nucleic acid comprising
immobilizing a nucleic acid that comprises a plurality of tandemly arranged identical nucleic acids onto a solid support, flowing oligonucleotides of known sequence over the immobilized nucleic acid, and detecting hybridization of the oligonucleotides to the immobilized nucleic acid by changes in length of the immobilized nucleic acid.
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29. (canceled)
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30. A method for sequencing a nucleic acid comprising
immobilizing a template nucleic acid on a solid support surface, wherein the template nucleic acid is hybridized to a plurality of primers and is bound to a plurality of polymerases, incorporating one or more known nucleotide triphosphates simultaneously at the 3′ - end of each primer, and
detecting the incorporation of the one or more known nucleotide triphosphates by a change in length of the template nucleic acid.
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31. A method for sequencing a nucleic acid comprising
immobilizing a plurality of nucleic acids that each comprises a plurality of tandemly arranged identical nucleic acids onto a solid support surface, performing a plurality of sequencing-by-synthesis reactions using the immobilized nucleic acids as templates, and detecting incorporation of nucleotides in the absence of fluorescence, wherein each of the plurality of template nucleic acids is bound to a separate region of the solid support surface that is contiguous and in fluid communication with all other regions on the solid support surface.
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32. A method for sequencing a nucleic acid comprising
immobilizing a plurality of nucleic acids that each comprises a plurality of tandemly arranged identical nucleic acids onto a solid support surface, performing a plurality of sequencing-by-synthesis reactions using the immobilized nucleic acids as templates, and detecting incorporation of nucleotides for each individual immobilized nucleic acid.
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33. (canceled)
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34. A method for sequencing a nucleic acid comprising
(a) immobilizing each of a plurality of template nucleic acids in separate non-overlapping regions of a solid support surface in a flow cell, each of the template nucleic acids comprising a plurality of tandemly arranged identical nucleic acid sequences and hybridized to a plurality of primers and bound to a plurality of polymerases, (b) introducing a plurality of known identical unlabeled nucleotide triphosphates into the flow cell, (c) for each individual template nucleic acid, detecting incorporation of one or more nucleotide triphosphates to the plurality of hybridized primers, (d) washing unincorporated nucleotide triphosphates from the flow cell, and (e) repeating steps (b) through (d) in the same flow cell using a different plurality of known nucleotide triphosphates, wherein all template nucleic acids within the flow cell are in fluid communication with each other.
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35. A method for sequencing a nucleic acid comprising
performing a plurality of sequencing-by-synthesis reactions using a plurality of immobilized template nucleic acids each comprising a plurality of tandemly arranged identical nucleic acid sequences, wherein nucleotide incorporation is detected in a non-fluorescent manner and sequences ranging from 600-1000 bases are determined from individual template nucleic acids.
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36. A method for sequencing a nucleic acid comprising
detecting incorporation of one or more known unlabeled nucleotide triphosphates to a plurality of primers hybridized to a template nucleic acid immobilized on a wall of a flow cell.
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37-38. -38. (canceled)
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39. A method for sequencing a nucleic acid comprising
fragmenting a nucleic acid to generate a target nucleic acid, individually amplifying a target nucleic acid to generate a template nucleic acid comprising tandemly arranged multiple copies of the target nucleic acid, and sequencing an individual template nucleic acid using unlabeled nucleotide triphosphates in the absence of fluorescence.
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40. A method for sequencing a nucleic acid comprising
individually sequencing a template nucleic acid that is immobilized on a solid support surface and is in fluid communication with other immobilized template nucleic acids using unlabeled nucleotide triphosphates.
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41. A method for generating a nucleic acid comprising
isothermally amplifying a circular target nucleic acid using natural nucleotide triphosphates for a first set of amplification reactions and modified nucleotide triphosphates for a second set of amplification reactions to generate a template nucleic acid, wherein the first set of amplification reactions occur prior to the second set of reactions.
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42-46. -46. (canceled)
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47. An article comprising
at least two polyethylene glycol populations immobilized to a solid support surface.
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48-56. -56. (canceled)
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57. An apparatus comprising
a flow cell having an inlet and an outlet, and a wall having immobilized thereon in separate non-overlapping regions each of a plurality of nucleic acids, and wherein the non-overlapping regions are in fluid communication with each other, and a CMOS imager.
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58-63. -63. (canceled)
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64. A method for measuring length comprising
detecting a bead tethered to a known location on a solid support, and determining a distance moved by the bead over time.
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65-69. -69. (canceled)
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70. A method for determining a position of an observable moiety, comprising
detecting signal from an observable moiety, and subtracting from said signal a local average background signal, wherein the observable moiety is one of a plurality of observable moieties tethered to a solid support.
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71-76. -76. (canceled)
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77. A method for determining movement of an observable moiety tethered to a solid support comprising
detecting a reference observable moiety and a test observable moiety, determining a level of movement of the reference observable moiety during a first time period to obtain a reference movement, and subtracting reference movement from a level of movement of the test observable moiety during the first time period, wherein the reference and test observable moieties are tethered to a solid support.
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78-81. -81. (canceled)
Specification