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    Method for testing nucleic acid sequence
    2.
    发明授权
    Method for testing nucleic acid sequence 失效
    核酸序列测试方法

    公开(公告)号：US06821734B2

    公开(公告)日：2004-11-23

    申请号：US10083340

    申请日：2002-02-27

    申请人： Hideki Kambara ,  Guohua Zhou ,  Kazunori Okano ,  Keiichi Nagai

    发明人： Hideki Kambara ,  Guohua Zhou ,  Kazunori Okano ,  Keiichi Nagai

    IPC分类号： C12Q168

    CPC分类号： C12Q1/682 ,  C12Q1/6827 ,  C12Q1/6858 ,  C12Q2565/537 ,  C12Q2561/125 ,  C12Q2531/125 ,  C12Q2525/301 ,  C12Q2565/301 ,  C12Q2565/519

    摘要： A method for examining nucleotide sequences of a sample includes adding a group of primers of multiple species to a solution containing the sample and simultaneously synthesizing complementary strands at each of the multiple regions containing the nucleotide sequences; designing the DNA probes with specific sequences elongate the complementary strands by the presence or absence of mutations in the nucleotide sequences, wherein the same number of such DNA probes and the nucleotide sequences are used for complementary strand synthesis; using the nucleotide sequences or their complementary sequences as a template to convert pyrophosphate produced during the elongation reaction to ATP which then reacts with chemiluminescent substrates to develop luminescence to be detected. According to the method, sensitivity is greatly increased by amplification of the amount of pyrophosphate produced in synthesis of the complementary strands without amplifying the copies of nucleotide sequences.

    摘要翻译： 检查样品的核苷酸序列的方法包括将一组多种引物添加到含有样品的溶液中，同时在含有该核苷酸序列的多个区域的每个区域合成互补链; 用特定序列设计DNA探针，通过核苷酸序列中存在或不存在突变来延长互补链，其中相同数量的这种DNA探针和核苷酸序列用于互补链合成; 使用核苷酸序列或其互补序列作为模板将在延伸反应期间产生的焦磷酸转化为ATP，然后与化学发光底物反应以产生待检测的发光。 根据该方法，在不扩增核苷酸序列的拷贝的情况下，通过扩增合成互补链中产生的焦磷酸盐的量来大大提高灵敏度。

    Method for determining nucleic acids base sequence and apparatus therefor
    3.
    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。

    DNA probe array
    5.
    发明授权
    DNA probe array 有权
    DNA探针阵列

    公开(公告)号：US06288220B1

    公开(公告)日：2001-09-11

    申请号：US09260543

    申请日：1999-03-02

    申请人： Hideki Kambara ,  Kazunori Okano

    发明人： Hideki Kambara ,  Kazunori Okano

    IPC分类号： C07H2104

    CPC分类号： C07H21/00 ,  B01J19/0046 ,  B01J2219/00459 ,  B01J2219/005 ,  B01J2219/0052 ,  B01J2219/00585 ,  B01J2219/00596 ,  B01J2219/00605 ,  B01J2219/0061 ,  B01J2219/00612 ,  B01J2219/00621 ,  B01J2219/00626 ,  B01J2219/00628 ,  B01J2219/0063 ,  B01J2219/00644 ,  B01J2219/00648 ,  B01J2219/00657 ,  B01J2219/00702 ,  B01J2219/00707 ,  B01J2219/00722 ,  C40B40/06 ,  Y10S436/805 ,  Y10S436/809

    摘要： There is used at least one probe array obtained by arraying particles having various probes, respectively, fixed thereon (probe particles) in a definite order in a holder. A plurality of capillaries or grooves packed with various kinds, respectively, of probe particles are arrayed in parallel, and one of particles contained in each capillary or groove is injected into another capillary or groove to produce a probe array in which the various kinds of probe particles are arrayed in a constant and definite order. Various fluorophore-labeled DNA's are measured at the same time by attaching various probes to particles, respectively, of different sizes. A probe array composed of various fixed DNA probes can easily be produced, and there can be provided a probe array for detecting various DNA's which is composed of various fixed arbitrary DNA probes.

    摘要翻译： 使用至少一个探针阵列，其通过将具有各种探针的颗粒分别在固定器上以确定的顺序分别固定在其上（探针颗粒）而获得。 多个分别装有探针颗粒的毛细管或凹槽平行地排列，并且将每个毛细管或凹槽中包含的一个颗粒注入另一个毛细管或凹槽中以产生探针阵列，其中各种探针 颗粒以恒定且明确的顺序排列。 通过将各种探针分别附着到不同尺寸的颗粒上同时测量各种荧光团标记的DNA。 由各种固定的DNA探针组成的探针阵列可以容易地生成，并且可以提供用于检测由各种固定的任意DNA探针组成的各种DNA的探针阵列。

    Method of analysis or assay for polynucleotides and analyzer or instrument for polynucleotides
    6.
    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。

    DNA measuring method
    7.
    发明授权
    DNA measuring method 失效
    DNA测定方法

    公开(公告)号：US5356776A

    公开(公告)日：1994-10-18

    申请号：US942470

    申请日：1992-09-09

    申请人： Hideki Kambara ,  Kazunori Okano ,  Satoshi Takahashi ,  Keiichi Nagai ,  Tetsuo Nishikawa

    发明人： Hideki Kambara ,  Kazunori Okano ,  Satoshi Takahashi ,  Keiichi Nagai ,  Tetsuo Nishikawa

    IPC分类号： C12M1/00 ,  C12Q1/68 ,  G01N27/447 ,  G01N33/53 ,  G01N33/58 ,  C12Q1/70

    CPC分类号： G01N27/44726 ,  C12Q1/68 ,  G01N27/447 ,  Y10T436/143333

    摘要： DNA molecule length can be measured with high precision and efficiency by 1) using such means as electrophoresis gel migration to orient a DNA molecule having a fluorescence label at both its termini into a straight line by its passing through a migration path having in a portion of it an area not more than several micrometers in diameter, detecting the fluorescence label at both the termini at a predetermined location and measuring the interval between the detection of the fluorescence coming from one terminus and that of the fluorescence from the other or by 2) a DNA molecule bound to a fluorescence label at one terminus and to a particle at the other being led as a whole by such means as electric field application into an aperture smaller in diameter than the particle, leaving the particle fixed at the mouth of the aperture to stretch the DNA molecule and detecting the fluorescence position to measure the distance between the bound particle and the bound fluorescence label.

    摘要翻译： 可以通过以下方式测量DNA分子长度：1）使用电泳凝胶迁移的方式，通过其经过一部分迁移路径将具有两端的荧光标记的DNA分子定向成直线 它是直径不超过几微米的区域，在预定位置的两个末端检测荧光标记，并测量从一个末端检测荧光和来自另一个末端的荧光检测之间的间隔，或者通过2）a 在一个末端与一个荧光标记结合的DNA分子和另一个的一个粒子整体通过电场施加到比颗粒直径更小的孔隙中，将颗粒固定在孔的口处 拉伸DNA分子并检测荧光位置以测量结合的颗粒和结合的荧光标记之间的距离。

    DNA probe array
    8.
    发明授权
    DNA probe array 有权
    DNA探针阵列

    公开(公告)号：US07364850B2

    公开(公告)日：2008-04-29

    申请号：US10865846

    申请日：2004-06-14

    申请人： Hideki Kambara ,  Kazunori Okano

    发明人： Hideki Kambara ,  Kazunori Okano

    IPC分类号： C12Q1/68 ,  C12M1/36 ,  G01N15/06

    CPC分类号： C07H21/00 ,  B01J19/0046 ,  B01J2219/00459 ,  B01J2219/005 ,  B01J2219/0052 ,  B01J2219/00585 ,  B01J2219/00596 ,  B01J2219/00605 ,  B01J2219/0061 ,  B01J2219/00612 ,  B01J2219/00621 ,  B01J2219/00626 ,  B01J2219/00628 ,  B01J2219/0063 ,  B01J2219/00644 ,  B01J2219/00648 ,  B01J2219/00657 ,  B01J2219/00702 ,  B01J2219/00707 ,  B01J2219/00722 ,  C40B40/06 ,  Y10S436/805 ,  Y10S436/809

    摘要： There is used at least one probe array obtained by arraying particles having various probes, respectively, fixed thereon (probe particles) in a definite order in a holder. A plurality of capillaries or grooves packed with various kinds, respectively, of probe particles are arrayed in parallel, and one of particles contained in each capillary or groove is injected into another capillary or groove to produce a probe array in which the various kinds of probe particles are arrayed in a constant and definite order. Various fluorophore-labeled DNA's are measured at the same time by attaching various probes to particles, respectively, of different sizes. A probe array composed of various fixed DNA probes can easily be produced, and there can be provided a probe array for detecting various DNA's which is composed of various fixed arbitrary DNA probes.

    摘要翻译： 使用至少一个探针阵列，其通过将具有各种探针的颗粒分别在固定器上以确定的顺序分别固定在其上（探针颗粒）而获得。 多个分别装有探针颗粒的毛细管或凹槽平行地排列，并且将每个毛细管或凹槽中包含的一个颗粒注入另一个毛细管或凹槽中以产生探针阵列，其中各种探针 颗粒以恒定且明确的顺序排列。 通过将各种探针分别附着到不同尺寸的颗粒上同时测量各种荧光团标记的DNA。 由各种固定的DNA探针组成的探针阵列可以容易地生成，并且可以提供用于检测由各种固定的任意DNA探针组成的各种DNA的探针阵列。

    Apparatus for determining base sequence of nucleic acid
    10.
    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。