Apparatus for determining base sequence of nucleic acid
First Claim
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1. An apparatus for determining a base sequence of a template DNA comprising:
- a cell in which a complementary strand extension reaction using a polymerase is performed for extending a hybridized primer hybridized with a template DNA or extending an extended primer produced by said complementary strand extension reaction, 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;
a laser which excites at least one fluorophore label included in said chemical modification of said chemically modified nucleotide incorporated to 3′
-terminus of said hybridized primer or said extended primer;
a lens which collects fluorescence emitted from said fluorophore label(s);
filters, each of which selects a wavelength of said fluorescence;
a photodetector which detects said fluorescence selected by said filters;
an analyzer which determines a kind of a base of said chemically modified nucleotide incorporated to 3′
-terminus of said hybridized primer or said extended primer, based on an output of said photodetector; and
a light source which emits a light for 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;
wherein, by repeating said complementary strand extension reaction for extending said hybridized primer or said extended primer and releasing said chemical modification from said chemically modified nucleotide incorporated to 3′
-terminus of said hybridized primer or said extended primer, said base sequence of said template DNA is determined one base by one base sequentially form the kinds of bases of said chemically modified nucleotides incorporated to said extended primer by said complementary strand extension reaction.
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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.
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Citations
9 Claims
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1. An apparatus for determining a base sequence of a template DNA comprising:
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a cell in which a complementary strand extension reaction using a polymerase is performed for extending a hybridized primer hybridized with a template DNA or extending an extended primer produced by said complementary strand extension reaction, 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;
a laser which excites at least one fluorophore label included in said chemical modification of said chemically modified nucleotide incorporated to 3′
-terminus of said hybridized primer or said extended primer;
a lens which collects fluorescence emitted from said fluorophore label(s);
filters, each of which selects a wavelength of said fluorescence;
a photodetector which detects said fluorescence selected by said filters;
an analyzer which determines a kind of a base of said chemically modified nucleotide incorporated to 3′
-terminus of said hybridized primer or said extended primer, based on an output of said photodetector; and
a light source which emits a light for 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;
wherein, by repeating said complementary strand extension reaction for extending said hybridized primer or said extended primer and releasing said chemical modification from said chemically modified nucleotide incorporated to 3′
-terminus of said hybridized primer or said extended primer, said base sequence of said template DNA is determined one base by one base sequentially form 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, 7, 8)
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9. An apparatus for determining a base sequence of a template DNA comprising:
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a cell which holds a template DNA hybridized with a primer, wherein a buffer solution including four kinds of caged nucleotides flows into said cell, and wherein a complementary strand extension reaction using a polymerase is performed for extending said hybridized primer or an extended primer produced by said complementary strand extension reaction, 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, 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 3′
-terminus of said hybridized primer or said extended primer;
a laser which excites said fluorophore label(s) of said caged nucleotide incorporated to 3′
-terminus of said hybridized primer or said extended primer;
a lens which collects fluorescence emitted from said fluorophore label(s);
filters, each of which selects a wavelength of said fluorescence;
a photodetector which detects said fluorescence selected by said filters;
an analyzer which determines a kind of a base of said caged nucleotide incorporated to 3′
-terminus of said hybridized primer or said extended primer, based on an output of said photodetector; and
a light source which emits a light for 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;
wherein by repeating said complementary strand extension reaction for extending said hybridized primer or said extended primer and releasing said fluorophore label(s) from said caged nucleotides incorporated to 3′
-terminus of said hybridized primer or said extended primer, said base sequence of said template DNA is determined one base by one base sequentially form the kinds of bases of said caged nucleotides incorporated to said extended primer by said complementary strand extension reaction.
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Specification
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Current AssigneeHitachi, Ltd.
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Original AssigneeHitachi, Ltd.
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InventorsAnazawa, Takashi, Kambara, Hideki, Uematsu, Chihiro, Okano, Kazunori
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Primary Examiner(s)Riley, Jezia
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Application NumberUS09/567,270Time in Patent Office392 DaysField of Search435/6, 435/91.1, 435/91.2, 536/22.1, 536/23.1, 536/24.3, 536/24.33, 536/25.3US Class Current435/6.12CPC Class CodesC12Q 1/6869 Methods for sequencingC12Q 2523/319 Photocleavage, photolysis, ...C12Q 2537/149 Sequential reactionsG01N 21/6458 Fluorescence microscopy flu...G01N 21/648 using evanescent coupling o...G01N 21/6486 Measuring fluorescence of b...
