Method for determining nucleic acids base sequence and apparatus therefor
First Claim
1. A method determining a base sequence of a template DNA comprising the steps of:
- (a) hybridizing a primer with a template DNA;
(b) performing a complementary strand extension reaction using a polymerase for extending said hybridized primer or an extended primer produced by repeating said step (b) to following step (d), by incorporating a single chemically modified nucleotide of four kinds of chemically modified nucleotides to 3'"'"'-terminus of said hybridized primer or said extended primer, in the presence of said four kinds of chemically modified nucleotides, wherein said single chemically modified nucleotide is complementary with a base sequence of said template DNA, and each of said four kinds of chemically modified nucleotides has a chemical modification for preventing a continuous progress of said complementary strand extension reaction, after incorporating said single chemically modified nucleotide to 3'"'"'-terminus of said hybridized primer or said extended primer;
(c) detecting said single chemically modified nucleotide incorporated to 3'"'"'-terminus of said hybridized primer or said extended primer, and determining a kind of a base of said single chemically modified nucleotide incorporated to 3'"'"'-terminus of said hybridized primer or said extended primer;
(d) carrying out a chemical reaction for changing a chemical structure of said single chemically modification nucleotide incorporated to 3'"'"'-terminus of said hybridized primer or said extended primer, in order to bring about a state at which said complementary strand extension reaction for extending said hybridized primer or said extended primer can proceed; and
(e) repeating said step (b) to said step (d), and determining said base sequence of said template DNA one base by one base sequentially based on the kinds of bases of said chemically modified nucleotides incorporated to said extended primer by said complementary strand extension reaction.
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Accused Products
Abstract
A single molecule of single-stranded sample DNA (7) having a bead (5) at one end and a magnetic bead (6) at the other end is extended and fixed in the field of view of a fluorescent microscope by using a magnetic force (11) and a laser trap (3), and a primer (8) is bonded thereto, followed by elongation reaction (10) using polymerase. Only a single chemically modified nucleotide (9) labeled with at least one fluorophore which varies depending on the kind of the base is incorporated. Only the single fluorophore incorporated is measured as a fluorescence-microscopic image by evanescent irradiation (13) with exciting laser beams, and the kind of the base is determined from the kind of the fluorophore. The fluorophore labeling the nucleotide incorporated is released by evanescent irradiation (13) with ultraviolet laser beams (2), and the next nucleotide is incorporated. DNA sequencing is carried out by repeating the above procedure. The base sequence determination can be carried out by using the single DNA molecule, so that a DNA base sequence of hundreds kilos or more bases can be efficiently determined.
223 Citations
12 Claims
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1. A method determining a base sequence of a template DNA comprising the steps of:
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(a) hybridizing a primer with a template DNA; (b) performing a complementary strand extension reaction using a polymerase for extending said hybridized primer or an extended primer produced by repeating said step (b) to following step (d), by incorporating a single chemically modified nucleotide of four kinds of chemically modified nucleotides to 3'"'"'-terminus of said hybridized primer or said extended primer, in the presence of said four kinds of chemically modified nucleotides, wherein said single chemically modified nucleotide is complementary with a base sequence of said template DNA, and each of said four kinds of chemically modified nucleotides has a chemical modification for preventing a continuous progress of said complementary strand extension reaction, after incorporating said single chemically modified nucleotide to 3'"'"'-terminus of said hybridized primer or said extended primer; (c) detecting said single chemically modified nucleotide incorporated to 3'"'"'-terminus of said hybridized primer or said extended primer, and determining a kind of a base of said single chemically modified nucleotide incorporated to 3'"'"'-terminus of said hybridized primer or said extended primer; (d) carrying out a chemical reaction for changing a chemical structure of said single chemically modification nucleotide incorporated to 3'"'"'-terminus of said hybridized primer or said extended primer, in order to bring about a state at which said complementary strand extension reaction for extending said hybridized primer or said extended primer can proceed; and (e) repeating said step (b) to said step (d), and determining said base sequence of said template DNA one base by one base sequentially based on the kinds of bases of said chemically modified nucleotides incorporated to said extended primer by said complementary strand extension reaction. - View Dependent Claims (2, 3, 4, 5, 6)
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7. A method determining a base sequence of a template DNA comprising the steps of:
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(a) hybridizing a primer with a template DNA; (b) performing a complementary strand extension reaction using a polymerase for extending said hybridized primer or an extended primer produced by repeating said step (b) to following step (d), by incorporating a single chemically modified nucleotide of four kinds of chemically modified nucleotides to 3'"'"'-terminus of said hybridized primer or said extended primer, in a flow of a buffer solution including said four kinds of chemically modified nucleotides, wherein said single chemically modified nucleotide is complementary with a base sequence of said template DNA, and each of said four kinds of chemically modified nucleotides has a chemical modification for preventing a continuous progress of said complementary strand extension reaction, after incorporating said single chemically modified nucleotide to 3'"'"'-terminus of said hybridized primer or said extended primer; (c) detecting said single chemically modified nucleotide incorporated to 3'"'"'-terminus of said hybridized primer or said extended primer, and determining a kind of a base of said single chemically modified nucleotide incorporated to 3'"'"'-terminus of said hybridized primer or said extended primer; (d) carrying out a chemical reaction for changing a chemical structure of said single chemically modification nucleotide incorporated to 3'"'"'-terminus of said hybridized primer or said extended primer, in order to bring about a state at which said complementary strand extension reaction for extending said hybridized primer or said extended primer can proceed; and (e) repeating said step (b) to said step (d), and determining said base sequence of said template DNA one base by one base sequentially based on the kinds of bases of said chemically modified nucleotides incorporated to said extended primer by said complementary strand extension reaction.
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8. A method of determining a base sequence of a template DNA comprising the steps of:
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(a) hybridizing a primer with a template DNA; (b) performing a complementary strand extension reaction using a polymerase for extending said hybridized primer or an extended primer produced by repeating said step (b) to following step (d), by incorporating a single caged nucleotide of four kinds of caged nucleotides to 3'"'"'-terminus of said hybridized primer or said extended primer, in the presence of said four kinds of caged nucleotides, wherein said single caged nucleotide is complementary with a base sequence of said template DNA, and each of four kinds of caged nucleotides has at least one fluorophore label, and a continuous progress of said complementary strand extension reaction is prevented after incorporating said single caged nucleotide to 3'"'"'-terminus of said hybridized primer or said extended primer; (c) exciting said fluorophore label(s) included in said single caged nucleotide incorporated to 3'"'"'-terminus of said hybridized primer or said extended primer, by laser irradiation to emit fluorescence from said fluorophore label(s), and detecting fluorescence to determine a kind of a base of said caged nucleotide incorporated to 3'"'"'-terminus of said hybridized primer or said extended primer, and determining a kind of a base of said single caged nucleotide incorporated to 3'"'"'-terminus of said hybridized primer or said extended primer; (d) releasing said fluorophore label(s) from said caged nucleotide incorporated to 3'"'"'-terminus of said hybridized primer or said extended primer, in order to bring about a state at which said complementary strand extension reaction for extending said hybridized primer or said extended primer can proceed; and (e) repeating said step (b) to said step (d), and determining said base sequence of said template DNA one base by one base sequentially based on the kinds of bases of said caged nucleotides incorporated to said extended primer by said complementary strand extension reaction.
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9. A method determining a base sequence of a template DNA comprising the steps of:
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(a) hybridizing a primer with a template DNA; (b) performing a complementary strand extension reaction using a polymerase for extending said hybridized primer or an extended primer produced by repeating said step (b) to following step (d), by incorporating a single chemically modified nucleotide of four kinds of chemically modified nucleotides to 3'"'"'-terminus of said hybridized primer or said extended primer, in the presence of said four kinds of chemically modified nucleotides, wherein said single chemically modified nucleotide is complementary with a base sequence of said template DNA, and each of said four kinds of chemically modified nucleotides has a chemical modification for preventing a continuous progress of said complementary strand extension reaction, after incorporating said single caged nucleotide to 3'"'"'-terminus of said hybridized primer or said extended primer; (c) detecting said single chemically modified nucleotide incorporated to 3'"'"'-terminus of said hybridized primer or said extended primer, and determining a kind of a base of said single chemically modified nucleotide incorporated to 3'"'"'-terminus of said hybridized primer or said extended primer; (d) releasing said chemical modification from said chemically modified nucleotide incorporated to 3'"'"'-terminus of said hybridized primer or said extended primer, in order to bring about a state at which said complementary strand extension reaction for extending said hybridized primer or said extended primer can proceed; and (e) repeating said step (b) to said step (d), and determining said base sequence of said template DNA one base by one base sequentially based on the kinds of bases of said chemically modified nucleotides incorporated to said extended primer by said complementary strand extension reaction.
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10. A method determining a base sequence of a template DNA comprising the steps of:
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(a) hybridizing a primer with a template DNA; (b) performing a complementary strand extension reaction using a polymerase for extending said hybridized primer or an extended primer produced by repeating said step (b) to following step (d), by incorporating a single chemically modified nucleotide of four kinds of chemically modified nucleotides to 3'"'"'-terminus of said hybridized primer or said extended primer, in a flow of a buffer solution including said four kinds of chemically modified nucleotides, wherein said single chemically modified nucleotide is complementary with a base sequence of said template DNA, and each of said four kinds of chemically modified nucleotides has a chemical modification for preventing a continuous progress of said complementary strand extension reaction, after incorporating said single chemically modified nucleotide to 3'"'"'-terminus of said hybridized primer or said extended primer; (c) detecting said single chemically modified nucleotide incorporated to 3'"'"'-terminus of said hybridized primer or said extended primer, and determining a kind of a base of said single chemically modified nucleotide incorporated to 3'"'"'-terminus of said hybridized primer or said extended primer; (d) releasing said chemical modification from said chemically modified nucleotide incorporated to 3'"'"'-terminus of said hybridized primer or said extended primer, in order to bring about a state at which said complementary strand extension reaction for extending said hybridized primer or said extended primer can proceed; and (e) repeating said step (b) to said step (d), and determining said base sequence of said template DNA one base by one base sequentially based on the kinds of bases of said chemically modified nucleotides incorporated to said extended primer by said complementary strand extension reaction. - View Dependent Claims (11)
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12. A method determining a base sequence of a template DNA comprising the steps of:
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(a) hybridizing a primer with a template DNA; (b) performing a complementary strand extension reaction using a polymerase for extending said hybridized primer or an extended primer produced by repeating said step (b) to following step (d), by incorporating a single caged nucleotide of four kinds of caged nucleotides to 3'"'"'-terminus of said hybridized primer or said extended primer, in a flow of a buffer solution including said four kinds of caged nucleotides, wherein said single caged nucleotide is complementary with a base sequence of said template DNA, and each of said four kinds of caged nucleotides has a at least one fluorophore label a continuous progress of said complementary strand extension reaction is prevented after incorporating said single caged nucleotide to 3'"'"'-terminus of said hybridized primer or said extended primer; (c) exciting said fluorophore label(s) included in said single caged nucleotide incorporated to 3'"'"'-terminus of said hybridized primer or said extended primer, by laser irradiation to emit fluorescence from said fluorophore label(s), and detecting fluorescence to determine a kind of a base of said caged nucleotide incorporated to 3'"'"'-terminus of said hybridized primer or said extended primer, and determining a kind of a base of said single caged nucleotide incorporated to 3'"'"'-terminus of said hybridized primer or said extended primer; (d) releasing said fluorophore label(s) from said caged nucleotide incorporated to 3'"'"'-terminus of said hybridized primer or said extended primer, in order to bring about a state at which said complementary strand extension reaction for extending said hybridized primer or said extended primer can proceed; and (e) repeating said step (b) to said step (d), and determining said base sequence of said template DNA one base by one base sequentially based on the kinds of bases of said caged nucleotides incorporated to said extended primer by said complementary strand extension reaction.
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Specification