Synthesis of four-color 3′-O-allyl modified photocleavable fluorescent nucleotides and related methods
First Claim
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1. A method for determining the sequence of a single-stranded DNA template comprising the following steps:
- (A) contacting the single-stranded DNA template with 9°
N polymerase in the presence of (i) a primer and (ii) four nucleotide analogues under conditions such that the 9°
N polymerase catalyzes DNA synthesis of a DNA extension product which has incorporated at its 3′
end, a nucleotide analogue complementary to, and base-paired with, a nucleotide residue which is not base-paired and is located at the 5′
end of the single-stranded DNA template to be sequenced,wherein each of the four nucleotide analogues comprises;
(a) a base selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil, (b) a deoxyribose, (c) a unique fluorophore cleavably attached to each base of the same type base, and (d) a removable chemical moiety bound to the 3′
-oxygen of the deoxyribose which blocks further DNA synthesis when it is incorporated into the extension product; and
wherein each fluorescent nucleotide analogue when incorporated into the DNA extension product is characterized by a predetermined fluorescence emission wavelength different from the fluorescence emission wavelength of each of the other three fluorescent nucleotide analogues when incorporated into the DNA extension product;
(B) removing nucleotide analogues not incorporated into the DNA extension product;
(C) determining the identity of the fluorescent nucleotide analogue incorporated into the DNA extension product based upon its characteristic fluorescence emission wavelength;
(D) treating the nucleotide analogue incorporated into the DNA extension product so as to remove the chemical moiety bound to the 3′
-oxygen of the deoxyribose and cleave the fluorophore from the base;
(E) repeating each of steps (A) to (D) to successively determine the identity of the nucleotide analogues incorporated at the 3′
end of each succeeding extension product so synthesized so as to thereby determine the sequence of the single-stranded DNA template.
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Abstract
This invention provides a process for making 3′-O-allyl-dGTP-PC-Biodopy-FL-510, 3′-O-allyl-dATP-PC-ROX, 3′-O-allyl-dCTP-PC-Bodipy-650 and 3′-O-allyl-dUTP-PC-R6G, and related methods.
116 Citations
28 Claims
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1. A method for determining the sequence of a single-stranded DNA template comprising the following steps:
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(A) contacting the single-stranded DNA template with 9°
N polymerase in the presence of (i) a primer and (ii) four nucleotide analogues under conditions such that the 9°
N polymerase catalyzes DNA synthesis of a DNA extension product which has incorporated at its 3′
end, a nucleotide analogue complementary to, and base-paired with, a nucleotide residue which is not base-paired and is located at the 5′
end of the single-stranded DNA template to be sequenced,wherein each of the four nucleotide analogues comprises;
(a) a base selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil, (b) a deoxyribose, (c) a unique fluorophore cleavably attached to each base of the same type base, and (d) a removable chemical moiety bound to the 3′
-oxygen of the deoxyribose which blocks further DNA synthesis when it is incorporated into the extension product; andwherein each fluorescent nucleotide analogue when incorporated into the DNA extension product is characterized by a predetermined fluorescence emission wavelength different from the fluorescence emission wavelength of each of the other three fluorescent nucleotide analogues when incorporated into the DNA extension product; (B) removing nucleotide analogues not incorporated into the DNA extension product; (C) determining the identity of the fluorescent nucleotide analogue incorporated into the DNA extension product based upon its characteristic fluorescence emission wavelength; (D) treating the nucleotide analogue incorporated into the DNA extension product so as to remove the chemical moiety bound to the 3′
-oxygen of the deoxyribose and cleave the fluorophore from the base;(E) repeating each of steps (A) to (D) to successively determine the identity of the nucleotide analogues incorporated at the 3′
end of each succeeding extension product so synthesized so as to thereby determine the sequence of the single-stranded DNA template. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 28)
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15. A method for determining the sequence of a single-stranded RNA template comprising the following steps:
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(A) contacting the single-stranded RNA template with an RNA polymerase in the presence of (i) a primer and (ii) four nucleotide analogues under conditions such that the RNA polymerase catalyzes RNA synthesis of an RNA extension product which has incorporated at its 3′
end, a nucleotide analogue complementary to, and base-paired with, a nucleotide residue which is not base-paired and is located at the 5′
end of the single-stranded RNA template to be sequenced,wherein each of the four nucleotide analogues comprises;
(a) a base selected from the group consisting of adenine, guanine, cytosine, and uracil, (b) a ribose, (c) a unique fluorophore cleavably attached to each base of the same type base, and (d) a removable chemical moiety bound to the 3′
-oxygen of the ribose which blocks further RNA synthesis when it is incorporated into the extension product; andwherein each fluorescent nucleotide analogue when incorporated into the RNA extension product is characterized by a predetermined fluorescence emission wavelength different from the fluorescence emission wavelength of each of the other three fluorescent nucleotide analogues when incorporated into the RNA extension product; (B) removing nucleotide analogues not incorporated into the RNA extension product; (C) determining the identity of the fluorescent nucleotide analogue incorporated into the RNA extension product based upon its characteristic fluorescence emission wavelength; (D) treating the nucleotide analogue incorporated into the RNA extension product so as to remove the chemical moiety bound to the 3′
-oxygen of the ribose and cleave the fluorophore from the base;(E) repeating each of steps (A) to (D) to successively determine the identity of the nucleotide analogues incorporated at the 3′
end of each succeeding extension product so synthesized so as to thereby determine the sequence of the single-stranded RNA template. - View Dependent Claims (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)
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Specification
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Current AssigneeTrustees Of Columbia University In The City Of New York (Columbia University)
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Original AssigneeTrustees Of Columbia University In The City Of New York (Columbia University)
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InventorsJu, Jingyue, Meng, Qinglin, Kim, Dae H., Bi, Lanrong, Bai, Xiaopeng, Turro, Nicholas J.
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Primary Examiner(s)Riley, Jezia
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Application NumberUS13/186,353Publication NumberTime in Patent Office1,666 DaysField of Search435/6.1, 435/91.1, 435/91.2, 536/26.6US Class Current1/1CPC Class CodesC07H 1/04 Introducing polyphosphoric ...C07H 19/067 with ribosyl as the sacchar...C07H 19/10 with the saccharide radical...C07H 19/14 Pyrrolo-pyrimidine radicalsC12Q 1/6806 Preparing nucleic acids for...C12Q 1/6869 Methods for sequencingC12Q 1/6874 involving nucleic acid arra...C12Q 2521/101 DNA polymeraseC12Q 2525/117 incorporating modified baseC12Q 2525/186 incorporating a non-extenda...C12Q 2563/107 fluorescence
