Computer-based methods and systems for sequencing of individual nucleic acid molecules
First Claim
1. A method for imaging a single labeled nucleotide on an individual double stranded nucleic acid molecule, comprising:
- (a) nicking a double stranded nucleic acid molecule elongated and fixed onto a surface so that the double stranded nucleic acid molecule remains accessible for enzymatic reactions with enzymes for the addition of a labeled nucleotide creating a nicked strand;
(b) enzymatically adding a single nucleotide comprising a label; and
(c) imaging the added label.
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Abstract
The present invention also relates to single molecule optical sequencing methods and systems for determining the nucleotide sequence of individual double stranded nucleic acid molecules elongated and fixed to a solid-surface by nicking the nucleic acid molecule, enzymatically adding labeled nucleotides and imaging the labeled nucleotides. The present invention also relates to methods and systems of single molecule optical sequencing using primer extension comprising the steps of elongating and fixing the nucleic acid molecule annealed with or to be annealed with at least one primer on a surface so that the nucleic acid remains accessible for enzymatic reactions with enzymes for the addition of labeled nucleotides; exposing the nucleic acid molecule annealed with at least one primer to a polymerase and dideoxy nucleotide; exposing the nucleic acid molecule to a polymerase and nucleotides including labeled nucleotides to produce a primer extension nucleic acid molecule; and imaging the labeled primer extension molecule to produce an image. The invention further relates to methods of imaging single or multiple labeled nucleotides enzymatically added to nicked individual double stranded nucleic acid molecules. The invention also provides a method of analysis of the images of labeled nucleotides or primer extension products using Bayesian estimation to determine the sequence of the nucleic acid molecules.
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Citations
51 Claims
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1. A method for imaging a single labeled nucleotide on an individual double stranded nucleic acid molecule, comprising:
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(a) nicking a double stranded nucleic acid molecule elongated and fixed onto a surface so that the double stranded nucleic acid molecule remains accessible for enzymatic reactions with enzymes for the addition of a labeled nucleotide creating a nicked strand;
(b) enzymatically adding a single nucleotide comprising a label; and
(c) imaging the added label. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 33)
(a) modifying the label after imaging the label in order to visualize the subsequently added labeled nucleotides; and
(b) repeating the steps of enzymatically adding a nucleotide comprising a label, imaging the label, and modifying the label to image multiple, consecutively added nucleotides.
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12. The method of claim 11, further comprising enzymatically displacing the nicked strand of the nucleic acid molecule.
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13. The method of claim 12, in which enzymatically displacing the nicked strand is performed using the Klenow fragment of DNA Polymerase I.
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14. The method of claim 11, further comprising enzymatically opening the nicked sites on the double stranded nucleic acid molecule.
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15. The method of claim 14, in which the step of opening the nicked sites is performed by an enzyme having 5′
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exonuclease activity.
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16. The method of claim 15, in which the enzyme having 5′
- -31 exonuclease activity is DNA Polymerase I or T7 exonuclease.
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17. The method of claim 11, in which the label is photolabile.
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18. The method of claim 11, in which the step of modifying the label after imaging is performed by photobleaching or photolysis.
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33. The method of claim 3, in which the step of modifying the label after imaging is performed by photobleaching or photolysis.
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19. A method for determining the nucleotide sequence of an individual double stranded nucleic acid molecule, comprising:
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(a) nicking a double stranded nucleic acid molecule elongated and fixed onto a surface so that the double stranded nucleic acid molecule remains accessible for enzymatic reactions with enzymes for the addition of labeled nucleotides forming a 3′
terminus to create an extended strand;
(b) extending the strand by adding at least one nucleotide comprising a label to the 3′
terminus of the nicked site; and
(c) imaging the added label. - View Dependent Claims (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34)
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35. A system for determining the nucleotide sequence of an individual double stranded nucleic acid molecule, comprising:
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(a) the double stranded nucleic acid molecule elongated and fixed onto a surface so that the nucleic acid molecule remains accessible for enzymatic reactions and/or hybridization reactions;
(b) a polymerase fixed on the surface;
(c) nucleotides comprising a label fixed on the surface; and
(d) a device for imaging the label to produce an image. - View Dependent Claims (36, 37, 38, 39, 40, 41, 42, 43, 44)
(a) accumulating signals of an addition site of the image;
(b) filtering the signals according to fluorescence intensity;
(c) correlating the signals with the backbone of the nucleic acid molecule;
(d) tabulating addition sites of the image using Bayesian inference estimation of the signals; and
(e) aligning and assembling the addition sites to determine a nucleotide addition.
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45. A method of determining the nucleotide sequence of an individual nucleic acid molecule, comprising:
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(a) exposing a nucleic acid molecule annealed with at least one primer elongated and fixed onto a surface so that the nucleic acid molecule remains accessible for enzymatic reactions with enzymes for the addition of labeled nucleotides to a polymerase and dideoxy nucleotides comprising a base and a label; and
(b) imaging the labeled nucleotides added onto the primer;
to determine the nucleotide sequence of the nucleic acid molecule by the addition of the labeled dideoxy nucleotide.
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46. A method of determining the nucleotide sequence of an individual nucleic acid molecule, comprising:
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(a) exposing a nucleic acid molecule annealed with at least one primer elongated and fixed onto a surface so that the nucleic acid molecule remains accessible for enzymatic reactions with enzymes for the addition of a labeled nucleotide to a polymerase and nucleotides comprising a base and a label; and
(b) imaging the labeled nucleotide added onto the primers;
to determine the nucleotide sequence of the nucleic acid molecule by the addition of the labeled dideoxy nucleotide.
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47. A method of determining the nucleotide sequence of an individual-nucleic acid molecule, comprising:
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(a) exposing a nucleic acid molecule annealed with at least one primer elongated and fixed onto a surface so that the nucleic acid molecule remains accessible for enzymatic reactions with enzymes for the addition of labeled nucleotides to a polymerase and dideoxy nucleotides;
(b) exposing the nucleic acid molecule annealed with at least one primer to a polymerase and nucleotides including nucleotides comprising a label to produce a labeled primer extension nucleic acid molecule; and
(c) imaging the labeled primer extension nucleic acid molecule to produce an image;
to determine the nucleotide sequence of the nucleic acid molecule by the absence of a primer extension product corresponding to the dideoxy nucleotides used in step (a). - View Dependent Claims (50)
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48. A method of determining the nucleotide sequence of an individual nucleic acid molecule, comprising:
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(a) elongating and fixing the nucleic acid molecule onto a surface so that the nucleic acid molecule remains accessible for enzymatic reactions with enzymes for the addition of labeled nucleotides;
(b) annealing at least one primer to the elongated and fixed nucleic acid molecule;
(c) exposing the nucleic acid molecule annealed with a primer to a polymerase and dideoxy nucleotides;
(d) exposing the nucleic acid molecule to a polymerase and nucleotides including nucleotides comprising a label to produce at least one labeled primer extension nucleic acid molecule; and
(e) imaging the labeled primer extension nucleic acid molecule to produce an image;
to determine the nucleotide sequence of the nucleic acid molecule by the absence of a primer extension product corresponding to the dideoxy nucleotides used in step (c).
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49. A system for determining the nucleotide sequence of an individual nucleic acid molecule, comprising:
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(a) an elongated and fixed nucleic acid molecule on a surface so that the nucleic acid molecules remain accessible for enzymatic reactions with enzymes for the addition of labeled nucleotides;
(b) at least one primer annealed to the nucleic acid molecule;
(c) a polymerase enzyme fixed on the surface to produce a primer extension product;
(d) dideoxy nucleotides fixed on the surface;
(e) nucleotides comprising a label fixed on the surface; and
(f) a device for imaging the elongated and fixed nucleic acid molecule to detect the presence of labeled nucleotides in the primer extension product to produce an image;
whereby the absence of the image of the primer extension product for a particular dideoxy nucleotide corresponds to the nucleotide sequence at one position of the nucleic acid molecule.
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51. A method for imaging multiple labeled nucleotides on an individual double stranded nucleic acid molecule, comprising:
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(a) nicking a nucleic acid molecule elongated and fixed onto a surface so that the double stranded nucleic acid molecule remains accessible for enzymatic reactions with enzymes for the addition of labeled nucleotides;
(b) enzymatically adding multiple nucleotides comprising at least four bases and at least four labels; and
(c) simultaneously imaging the added labels.
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Specification