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    Method for determining nucleic acids base sequence and apparatus therefor
    1.
    发明授权
    Method for determining nucleic acids base sequence and apparatus therefor 失效
    确定核酸碱基序列的方法及其装置

    公开(公告)号：US6136543A

    公开(公告)日：2000-10-24

    申请号：US355567

    申请日：1999-07-30

    申请人： Takashi Anazawa ,  Kazunori Okano ,  Chihiro Uematsu ,  Hideki Kambara

    发明人： Takashi Anazawa ,  Kazunori Okano ,  Chihiro Uematsu ,  Hideki Kambara

    IPC分类号： C12Q1/68 ,  C07H19/00 ,  C07H21/00 ,  C07H21/04 ,  C12P19/34

    CPC分类号： C12Q1/6869

    摘要： 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.

    摘要翻译： PCT No.PCT / JP97 / 00239 Sec。 371日期1999年7月30日第 102（e）1999年7月30日PCT PCT 1997年1月31日PCT公布。 公开号WO98 / 33939 日期1998年8月6日在一端具有珠（5）和另一端的磁珠（6）的单分子单链样品DNA（7）在荧光显微镜的视场中延伸固定 通过使用磁力（11）和激光捕获器（3），并将引物（8）与其结合，然后使用聚合酶进行伸长反应（10）。 只有一个化学修饰的核苷酸（9）被至少一个根据碱的种类而变化的荧光团标记。 通过用激发激光束的ev逝照射（13）测量所掺入的单个荧光团作为荧光显微镜图像，并且根据荧光团的种类确定碱基的种类。 标记所结合核苷酸的荧光团通过用紫外激光束（2）的ev逝照射（13）释放，并且并入下一个核苷酸。 通过重复上述步骤进行DNA测序。 碱基序列的测定可以通过使用单个DNA分子进行，从而可以有效地确定数百个碱基的DNA碱基序列。

    Apparatus for determining base sequence of nucleic acid
    2.
    发明授权
    Apparatus for determining base sequence of nucleic acid 失效
    用于确定核酸碱基序列的装置

    公开(公告)号：US06242193B1

    公开(公告)日：2001-06-05

    申请号：US09567270

    申请日：2000-05-09

    申请人： Takashi Anazawa ,  Kazunori Okano ,  Chihiro Uematsu ,  Hideki Kambara

    发明人： Takashi Anazawa ,  Kazunori Okano ,  Chihiro Uematsu ,  Hideki Kambara

    IPC分类号： C12Q168

    CPC分类号： G01N21/6458 ,  C12Q1/6869 ,  G01N21/648 ,  G01N21/6486 ,  C12Q2537/149 ,  C12Q2523/319

    摘要： 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.

    摘要翻译： 在一端具有珠（5）和另一端的磁珠（6）的单分子单链样品DNA（7）在荧光显微镜的视场中通过使用磁力 （11）和激光捕获器（3），并将引物（8）与其结合，然后使用聚合酶进行伸长反应（10）。 只有一个化学修饰的核苷酸（9）被至少一个根据碱的种类而变化的荧光团标记。 通过用激发激光束的ev逝照射（13）测量所掺入的单个荧光团作为荧光显微镜图像，并且根据荧光团的种类确定碱基的种类。 标记所结合核苷酸的荧光团通过用紫外激光束（2）的ev逝照射（13）释放，并且并入下一个核苷酸。 通过重复上述步骤进行DNA测序。 碱基序列的测定可以通过使用单个DNA分子进行，从而可以有效地确定数百个碱基的DNA碱基序列。

    Method of analysis or assay for polynucleotides and analyzer or instrument for polynucleotides
    4.
    发明授权
    Method of analysis or assay for polynucleotides and analyzer or instrument for polynucleotides 失效
    多核苷酸分析或测定方法，多核苷酸分析仪或仪器

    公开(公告)号：US5861252A

    公开(公告)日：1999-01-19

    申请号：US758220

    申请日：1996-11-27

    申请人： Hideki Kambara ,  Kazunori Okano ,  Chihiro Uematsu

    发明人： Hideki Kambara ,  Kazunori Okano ,  Chihiro Uematsu

    IPC分类号： G01N33/50 ,  C07H21/04 ,  C12M1/00 ,  C12N15/09 ,  C12Q1/44 ,  C12Q1/48 ,  C12Q1/68 ,  G01N27/447 ,  G06F17/30 ,  C07K3/14 ,  C12P19/34

    CPC分类号： C12Q1/6855 ,  C12Q1/683

    摘要： A method of analysis or assay for nucleotides comprises: (1) a step of digesting DNA with a restriction enzyme; (2) a step of discriminating a difference in sequences of the DNA fragments obtained in step (1) above around the 3' termini thereof with a DNA probe and extending the DNA probe by a complementary strand synthesis to fractionate the DNA fragments into groups; and, (3) a step of measuring lengths of the DNA fragments which belong to said groups, or length of the DNA probe extended by said complementary strand extension reaction; wherein the thus measured lengths obtained for every sequence of the bases of the DNA fragments around the 3' termini thereof are employed as fingerprints.

    摘要翻译： 核苷酸的分析或测定方法包括：（1）用限制酶消化DNA的步骤; （2）通过DNA探针区分3'末端的步骤（1）中获得的DNA片段的序列差异的步骤，并通过互补链合成扩增DNA探针，将DNA片段分离成组; 和（3）测量属于所述组的DNA片段的长度或通过所述互补链延伸反应延伸的DNA探针的长度的步骤; 其中将由此测量的长度为其3'末端周围的DNA片段的碱基的每个序列获得的长度用作指纹。

    Method for assaying DNA fragments in mixture
    6.
    发明授权
    Method for assaying DNA fragments in mixture 失效
    测定混合物中DNA片段的方法

    公开(公告)号：US06225064B1

    公开(公告)日：2001-05-01

    申请号：US09413814

    申请日：1999-10-07

    申请人： Chihiro Uematsu ,  Hideki Kambara ,  Kazunori Okano

    发明人： Chihiro Uematsu ,  Hideki Kambara ,  Kazunori Okano

    IPC分类号： C12Q168

    CPC分类号： C12Q1/6809 ,  C12Q1/6855 ,  C12Q2525/173

    摘要： The inventive method for assaying DNA fragments in mixture comprises step 1 of ligating different oligomers hybridizable to primers of the same melting temperature and the same length to individual groups of DNA fragments in a set of DNA fragments; step 2 of mixing together the groups of DNA fragments ligated with the oligomers; step 3 of simultaneous PCR of the groups of DNA fragments ligated with the oligomers in one receptacle by using the primers being complementary to the oligomers and corresponding to the individual groups; and step 4 of detecting PCR amplified DNA fragments; characterized in that the method enables the comparison of plural samples under no influence of PCR reproducibility.

    摘要翻译： 用于测定混合物中DNA片段的本发明方法包括连接不同寡聚物的步骤，所述寡聚物与一组DNA片段中的DNA片段的相同融合温度和相同长度的引物可混合;步骤2将DNA片段组合在一起 与寡聚体连接;步骤3，通过使用与低聚物互补并对应于各个基团的引物，在一个容器中与寡聚物连接的DNA片段组同时PCR; 检测PCR扩增的DNA片段; 其特征在于，该方法能够在不影响PCR再现性的情况下比较多个样品。

    Fractionation method for nucleotide fragments
    7.
    发明授权
    Fractionation method for nucleotide fragments 失效
    核苷酸片段的分级方法

    公开(公告)号：US5817464A

    公开(公告)日：1998-10-06

    申请号：US748900

    申请日：1996-11-15

    申请人： Hideki Kambara ,  Kazunori Okano ,  Chihiro Uematsu

    发明人： Hideki Kambara ,  Kazunori Okano ,  Chihiro Uematsu

    IPC分类号： C12N15/09 ,  C12Q1/68 ,  G01N27/447 ,  G01N33/53 ,  G01N37/00 ,  C07H21/04 ,  C12P19/34 ,  C12Q1/70

    CPC分类号： G01N27/44743 ,  C12Q1/6837 ,  C12Q1/6874 ,  G01N27/447

    摘要： A fractionation method for DNA fragments according to the present invention comprises a first step of preparing a probe chip or a set of probe chips immobilizing independently a DNA probe having a first sequence part having a specific known sequence part together with a part of enzyme recognition sequence and a second sequence part composed of a combination of one to six bases adjacent to the first sequence part at 3' terminus, a second step of introducing a DNA oligomer composed of a part of enzyme recognition sequence and a sequence complementary to the known sequence part into the fragment termini of DNA fragments from restriction enzyme cleavage, and a third step of placing the probe chip or the set of probe chips in a solution containing the nucleotide fragments with the introduced DNA oligomer produced at the second step, for at least hybridization and the complementary strand extension of the DNA probe, whereby the DNA fragments are fractionated.

    摘要翻译： 根据本发明的DNA片段的分级方法包括制备探针芯片或一组探针芯片的第一步骤，该探针芯片独立地固定有具有特定已知序列部分的第一序列部分的DNA探针以及酶识别序列的一部分 第二序列部分由与3'末端的第一序列部分相邻的1至6个碱基的组合构成，第2步骤，引入由部分酶识别序列组成的DNA寡聚体和与已知序列部分互补的序列 进入限制酶切割的DNA片段的片段末端，以及第三步骤，将探针芯片或探针芯片组放置在含有第二步产生的引入的DNA寡聚体的核苷酸片段的溶液中，用于至少杂交和 DNA探针的互补链延伸，由此分离DNA片段。

    DNA fragment preparation method for gene expression profiling
    8.
    发明授权
    DNA fragment preparation method for gene expression profiling 有权
    用于基因表达谱的DNA片段制备方法

    公开(公告)号：US06203988B1

    公开(公告)日：2001-03-20

    申请号：US09228942

    申请日：1999-01-12

    申请人： Hideki Kambara ,  Chihiro Uematsu

    发明人： Hideki Kambara ,  Chihiro Uematsu

    IPC分类号： C12Q168

    CPC分类号： C12Q1/6858 ,  C12Q2565/537 ,  C12Q2525/191 ,  C12Q2525/173

    摘要： A DNA fragment preparation method for DNA analysis comprising, i) preparing a plurality of DNA fragments from a sample DNA, and ii) amplifying a specific DNA fragment by PCR, using a pair of primers which hybridize with terminus sequences of the DNA fragments, and a specific primer which hybridizes specifically with a base sequence of the specific DNA fragment at a position between a priming site of one of primer the pair of primers and a priming site of another primer of the pair of primers. The specific primer hybridizes specifically with a base sequence at a middle position of the specific DNA fragment. Products of PCR are separated, by electrophoresis, and signals from DNA fragments originated in a known genes and signals from DNA fragments originated in a unknown genes are displayed separately on a display.

    摘要翻译： 一种用于DNA分析的DNA片段制备方法，包括：i）从样品DNA制备多个DNA片段，以及ii）使用与DNA片段的末端序列杂交的一对引物扩增通过PCR扩增特定DNA片段，以及 特异性引物，其特异性与特异性DNA片段的碱基序列在引物对引物之一的引物位点与该对引物的另一引物的引物位点之间的位置杂交。 特异性引物与特定DNA片段的中间位置的碱基序列特异性杂交。 PCR产物通过电泳分离，来自已知基因的DNA片段的信号和源自未知基因的DNA片段的信号分别显示在显示器上。

    Method for analysis of nucleic acid and DNA primer sets for use therein
    9.
    发明授权
    Method for analysis of nucleic acid and DNA primer sets for use therein 失效
    用于分析核酸和DNA引物组的方法

    公开(公告)号：US5948615A

    公开(公告)日：1999-09-07

    申请号：US834385

    申请日：1997-04-16

    申请人： Chihiro Uematsu ,  Hideki Kambara

    发明人： Chihiro Uematsu ,  Hideki Kambara

    IPC分类号： C12N15/10 ,  C12Q1/68 ,  C07H21/02 ,  C12N15/00 ,  C12P19/34

    CPC分类号： C12N15/1096 ,  C12Q1/683 ,  C12Q1/6855

    摘要： The present invention comprises a method for analysis of a nucleic acid which comprises:(1) a step of digesting a double-stranded DNA sample with a plurality of restriction enzymes to obtain double-stranded DNA fragments;(2) a step of ligating a plurality of oligonucleotides to the double-stranded DNA fragments respectively at the both ends thereof;(3) a step of dispensing a solution containing the double-stranded DNA fragments into a plurality of tubes;(4) a step of adding DNA primers comprising combinations of DNA primers selected from each set of a plurality of DNA primer sets comprising a plurality of labeled primers having a base sequence complementary to the base sequence of oligonucleotide and a part or all of the base sequence contiguous to the base sequence complementary to the base sequence of the oligonucleotide and recognized by the restriction enzymes and a selective base sequence of 1 to 4 bases at the 3'-end thereof, to the respective tubes corresponding to the combinations and performing a complementary strand synthesis reaction of the region of the double-stranded DNA fragments between the base sequences recognized by the two restriction enzymes; and,(5) a step of subjecting the products obtained by the complementary strand synthesis reaction to electrophoresis to produce a large number of DNA fragments from the long double stranded DNA sample digested with restriction enzymes and obtain fingerprinting patterns therefrom which enables to inspect the long double-stranded DNA sample.

    摘要翻译： 本发明包括核酸分析方法，其包括：（1）用多种限制酶消化双链DNA样品以获得双链DNA片段的步骤; （2）在其两端分别将多个寡核苷酸与双链DNA片段连接的步骤; （3）将含有双链DNA片段的溶液分配到多个管中的步骤; （4）添加DNA引物的步骤，其包含选自每组多个DNA引物组的DNA引物的组合，所述多个DNA引物组包含多个具有与寡核苷酸的碱基序列互补的碱基序列的标记引物和碱基的一部分或全部 与寡核苷酸的碱基序列互补的碱基序列连续的序列，并且被限制性内切酶和在其3'末端具有1至4个碱基的选择性碱基序列连接到对应于组合的各个管，并进行互补 在两个限制酶识别的碱基序列之间的双链DNA片段的区域的链合成反应; 和（5）使由互补链合成反应得到的产物进行电泳，从用限制酶消化的长双链DNA样品中产生大量DNA片段的步骤，并从中获得指纹图案，其能够检查长 双链DNA样品。

    Method for assaying DNA fragments in mixture
    10.
    发明申请
    Method for assaying DNA fragments in mixture 失效
    测定混合物中DNA片段的方法

    公开(公告)号：US20050100910A1

    公开(公告)日：2005-05-12

    申请号：US10634795

    申请日：2003-08-06

    申请人： Chihiro Uematsu ,  Hideki Kambara ,  Kazunon Okano

    发明人： Chihiro Uematsu ,  Hideki Kambara ,  Kazunon Okano

    IPC分类号： C12N15/09 ,  C12Q1/68 ,  G01N33/53 ,  C12P19/34

    CPC分类号： C12Q1/6809 ,  C12Q1/6855 ,  C12Q2525/173

    摘要： The inventive method for assaying DNA fragments in mixture comprises step 1 of ligating different oligomers hybridizable to primers of the same melting temperature and the same length to individual groups of DNA fragments in a set of DNA fragments; step 2 of mixing together the groups of DNA fragments ligated with the oligomers; step 3 of simultaneous PCR of the groups of DNA fragments ligated with the oligomers in one receptacle by using the primers being complementary to the oligomers and corresponding to the individual groups; and step 4 of detecting PCR amplified DNA fragments; characterized in that the method enables the comparison of plural samples under no influence of PCR reproducibility.

    摘要翻译： 用于测定混合物中DNA片段的本发明方法包括步骤1，将不同的低聚物连接到一组DNA片段中与相同融合温度和相同长度的引物相同的DNA片段的单个组; 步骤2，将与低聚物连接的DNA片段组合在一起; 通过使用与寡聚体互补并对应于各个基团的引物，在一个容器中与低聚物连接的DNA片段组同时PCR的步骤3; 和检测PCR扩增的DNA片段的步骤4; 其特征在于，该方法能够在不影响PCR再现性的情况下比较多个样品。