DNA SEQUENCING BY SYNTHESIS USING MODIFIED NUCLEOTIDES AND NANOPORE DETECTION
First Claim
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1. A method for determining the nucleotide sequence of a single-stranded DNA comprising:
- (a) contacting the single-stranded DNA, wherein the single-stranded DNA is in an electrolyte solution in contact with a nanopore in a membrane and wherein the single-stranded DNA has a primer hybridized to a portion thereof, with a DNA polymerase and at least four deoxyribonucleotide polyphosphate (dNPP) analogues under conditions permitting the DNA polymerase to catalyze incorporation of one of the dNPP analogues into the primer if it is complementary to the nucleotide residue of the single-stranded DNA which is immediately 5′
to a nucleotide residue of the single-stranded DNA hybridized to the 3′
terminal nucleotide residue of the primer, so as to form a DNA extension product, wherein each of the four dNPP analogues has the structure;
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Abstract
This disclosure is related to a method of sequencing a single-stranded DNA using deoxynucleotide polyphosphate analogues and translocation of tags from incorporated deoxynucleotide polyphosphate analogues through a nanopore.
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Citations
65 Claims
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1. A method for determining the nucleotide sequence of a single-stranded DNA comprising:
(a) contacting the single-stranded DNA, wherein the single-stranded DNA is in an electrolyte solution in contact with a nanopore in a membrane and wherein the single-stranded DNA has a primer hybridized to a portion thereof, with a DNA polymerase and at least four deoxyribonucleotide polyphosphate (dNPP) analogues under conditions permitting the DNA polymerase to catalyze incorporation of one of the dNPP analogues into the primer if it is complementary to the nucleotide residue of the single-stranded DNA which is immediately 5′
to a nucleotide residue of the single-stranded DNA hybridized to the 3′
terminal nucleotide residue of the primer, so as to form a DNA extension product, wherein each of the four dNPP analogues has the structure;- View Dependent Claims (5, 6, 7, 9, 12, 13, 14, 21, 22, 23, 24, 25, 26, 27, 28)
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2. A method for determining the nucleotide sequence of a single-stranded DNA comprising:
(a) contacting the single-stranded DNA, wherein the single-stranded DNA is in an electrolyte solution in contact with a nanopore in a membrane and wherein the single-stranded DNA has a primer hybridized to a portion thereof, a DNA polymerase and a deoxyribonucleotide polyphosphate (dNPP) analogue under conditions permitting the DNA polymerase to catalyze incorporation of the dNPP analogue into the primer if it is complementary to the nucleotide residue of the single-stranded DNA which is immediately 5′
to a nucleotide residue of the single-stranded DNA hybridized to the 3′
terminal nucleotide residue of the primer, so as to form a DNA extension product, wherein the dNPP analogue has the structure;
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3. A method for determining the nucleotide sequence of a single-stranded DNA comprising:
(a) contacting the single-stranded DNA, wherein the single-stranded DNA is in an electrolyte solution in contact with a nanopore in a membrane and wherein the single-stranded DNA has a primer hybridized to a portion thereof, with a DNA polymerase and at least four deoxyribonucleotide polyphosphate (dNPP) analogues under conditions permitting the DNA polymerase to catalyze incorporation of one of the dNPP analogues into the primer if it is complementary to the nucleotide residue of the single-stranded DNA which is immediately 5′
to a nucleotide residue of the single-stranded DNA hybridized to the 3′
terminal nucleotide residue of the primer, so as to form a DNA extension product, wherein each of the four dNPP analogues has a structure chosen from the following;
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4. A method for determining the nucleotide sequence of a single-stranded DNA comprising:
(a) contacting the single-stranded DNA, wherein the single-stranded DNA is in an electrolyte solution in contact with a nanopore in a membrane, wherein the single-stranded DNA has a primer hybridized to a portion thereof, a DNA polymerase and a deoxyribonucleotide polyphosphate (dNPP) analogue under conditions permitting the DNA polymerase to catalyze incorporation of the dNPP analogue into the primer if it is complementary to the nucleotide residue of the single-stranded DNA which is immediately 5′
to a nucleotide residue of the single-stranded DNA hybridized to the 3′
terminal nucleotide residue of the primer, so as to form a DNA extension product, wherein the dNPP analogue has the structure;
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8. (canceled)
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10-11. -11. (canceled)
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15-20. -20. (canceled)
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29-43. -43. (canceled)
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44. A compound having the structure:
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45-52. -52. (canceled)
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53. A compound having the structure:
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54-62. -62. (canceled)
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63. A method for determining the nucleotide sequence of a single-stranded RNA comprising:
(a) contacting the single-stranded RNA, wherein the single-stranded RNA is in an electrolyte solution in contact with a nanopore in a membrane, wherein the single-stranded RNA has a primer hybridized to a portion thereof, with a RNA polymerase and at least four ribonucleotide polyphosphate (rNPP) analogues under conditions permitting the RNA polymerase to catalyze incorporation of one of the rNPP analogues into the primer if it is complementary to the nucleotide residue of the single-stranded RNA which is immediately 5′
to a nucleotide residue of the single-stranded RNA hybridized to the 3′
terminal nucleotide residue of the primer, so as to form a RNA extension product, wherein each of the four rNPP analogues has the structure;
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64. A method for determining the nucleotide sequence of a single-stranded RNA comprising:
(a) contacting the single-stranded RNA, wherein the single-stranded RNA is in an electrolyte solution in contact with a nanopore in a membrane and wherein the single-stranded RNA has a primer hybridized to a portion thereof, a RNA polymerase and a ribonucleotide polyphosphate (rNPP) analogue under conditions permitting the RNA polymerase to catalyze incorporation of the rNPP analogue into the primer if it is complementary to the nucleotide residue of the single-stranded RNA which is immediately 5′
to a nucleotide residue of the single-stranded RNA hybridized to the 3′
terminal nucleotide residue of the primer, so as to form a RNA extension product, wherein the rNPP analogue has the structure;
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65-86. -86. (canceled)
Specification