Nucleic acid sequencing with solid phase capturable terminators
First Claim
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1. A method of determining the sequence of a nucleic acid, said method comprising:
- (a) preparing a reaction mixture corresponding to each base of said nucleic acid, wherein each of said reaction mixtures comprises a family of enzymatically produced capturable primer extension products produced by combining said nucleic acid, polymerase, deoxynucleotides, oligonucleotide primer and capturable dideoxy chain terminator in a reaction mixture under conditions sufficient to produce said capturable primer extension products;
(b) contacting each of said reaction mixtures with a solid phase capable of capturing said primer extension products comprising said capturable chain terminator through interaction of moities present on said solid phase and said chain terminator to produce solid phase captured primer extension products;
(c) separating said solid phase captured primer extension products from said reaction mixture;
(d) releasing said primer extension products from said solid phase;
(e) size separating and detecting said released primer extension products to produce sequencing data; and
(f) determining the sequence of said nucleic acid from said sequencing data.
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Abstract
Methods of enzymatic nucleic acid sequencing are provided in which solid-phase capturable chain terminators are employed. In the subject methods, sequencing fragments are generated, where the fragments comprise capturable chain terminators. The fragments are then captured on a solid phase and separated from the remaining components of the sequencing reaction. The fragments are then released from the solid phase, size separated and detected to yield sequencing data from which the sequence of the nucleic acid is determined.
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34 Claims
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1. A method of determining the sequence of a nucleic acid, said method comprising:
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(a) preparing a reaction mixture corresponding to each base of said nucleic acid, wherein each of said reaction mixtures comprises a family of enzymatically produced capturable primer extension products produced by combining said nucleic acid, polymerase, deoxynucleotides, oligonucleotide primer and capturable dideoxy chain terminator in a reaction mixture under conditions sufficient to produce said capturable primer extension products; (b) contacting each of said reaction mixtures with a solid phase capable of capturing said primer extension products comprising said capturable chain terminator through interaction of moities present on said solid phase and said chain terminator to produce solid phase captured primer extension products; (c) separating said solid phase captured primer extension products from said reaction mixture; (d) releasing said primer extension products from said solid phase; (e) size separating and detecting said released primer extension products to produce sequencing data; and (f) determining the sequence of said nucleic acid from said sequencing data. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 19, 20, 21, 22, 23, 24, 25)
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12. A method of determining the sequence of a DNA, said method comprising:
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(a) preparing four different reaction mixtures corresponding to each base of said nucleic acid, wherein each of said reaction mixtures comprises a family of enzymatically produced fluorescently labeled primer extension products comprising a first member of a specific binding pair produced by combining said nucleic acid, polymerase, deoxynucleotides, fluorescently labeled oligonucleotide primer and dideoxynucleotide bonded to said first member of a specific binding pair member; (b) contacting each of said reaction mixtures with a solid phase comprising a second member of said specific binding pair whereby said first and second members bind and solid phase captured fluorescently labeled primer extension products are produced; (c) separating said solid phase captured fluorescently labeled primer extension products from said reaction mixture; (d) releasing said captured fluorescently labeled primer extension products from said solid phase; (e) size separating and detecting said released fluorescently labeled primer extension products to produce an electropherogram; and (f) determining the sequence of said nucleic acid from said electropherogram. - View Dependent Claims (13, 14, 15, 16, 17)
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18. A method of determining the sequence of a DNA, said method comprising:
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(a) preparing a first reaction mixture comprising enzymatically produced fluorescently labeled solid phase capturable primer extension products terminating in "A," wherein said first reaction mixture is prepared by combining said DNA with DNA polymerase, dATP, dGTP, dCTP, dTTP, energy transfer labeled oligonucleotide primer and biotinylated ddATP; (b) preparing a second reaction mixture comprising enzymatically produced fluorescently labeled solid phase capturable primer extension products terminating in "G" wherein said second reaction mixture is prepared by combining said DNA with DNA polymerase, dATP, dGTP, dCTP, dTTP, energy transfer labeled oligonucleotide primer and biotinylated ddGTP; (c) preparing a third reaction mixture comprising enzymatically produced fluorescently labeled solid phase capturable primer extension products terminating in "C," wherein said third reaction mixture is prepared by combining said DNA with DNA polymerase, dATP, dGTP, dCTP, dTTP, energy transfer labeled oligonucleotide primer and biotinylated ddCTP; (d) preparing a fourth reaction mixture comprising enzymatically produced fluorescently labeled solid phase capturable primer extension products terminating in "T," wherein said first reaction mixture is prepared by combining said DNA with DNA polymerase, dATP, dGTP, dCTP, dTTP, energy transfer labeled oligonucleotide primer and biotinylated ddTTP; (e) pooling said first, second, third and fourth reaction mixtures into a single reaction mixture; (f) contacting said reaction mixture with a solid phase comprising streptavidin to produce captured fluorescently labeled primer extension products; (g) separating said captured fluorescently labeled primer extension products from said reaction mixture; (h) releasing said captured fluorescently labeled primer extension products from said solid phase; (i) electrophoretically size separating said released fluorescently labeled primer extension products; (j) irradiating said size separated fluorescently labeled primer extension products; (k) detecting light emitted from said irradiated fluorescently labeled primer extension products; (l) producing an electropherogram from said detected light; and (m) determining the sequence of said DNA from said electropherogram.
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26. A kit for use in determining the sequence a nucleic acid, said kit comprising:
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at least one of labeled oligonucleotide primer and labeled dNTPs; and solid phase capturable chain terminator. - View Dependent Claims (27, 28, 29, 30, 31)
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32. A kit for use in determining the sequence a nucleic acid, said kit comprising:
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a plurality of distinguishably labeled oligonucleotide primers; a plurality of containers; solid phase capturable chain terminator; and a solid phase capable of capturing said chain terminator. - View Dependent Claims (33, 34)
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Specification