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    PCR primers and a method for deciding a base sequence thereof regarding adenylation
    2.
    发明授权
    PCR primers and a method for deciding a base sequence thereof regarding adenylation 失效
    PCR引物和用于确定其关于腺苷酸化的碱基序列的方法

    公开(公告)号：US06773901B2

    公开(公告)日：2004-08-10

    申请号：US10083616

    申请日：2002-02-27

    申请人： Chihiro Uematsu ,  Kazunori Okano ,  Takashi Irie

    发明人： Chihiro Uematsu ,  Kazunori Okano ,  Takashi Irie

    IPC分类号： C12P1934

    CPC分类号： C12Q1/6853 ,  C12Q2525/161

    摘要： Adenylation of a DNA fragment with a DNA polymerase occurs in the course of PCR, and thus two peaks are detected. To prevent the peak splitting, it is necessary to raise efficiency of adenylation a single peak to occur without changing reaction conditions. To this end, four types of PCR primers which, respectively, have an anchor sequence at 5′ terminus so that any of A, C, G or T is attached to at the 5′ terminus of the anchor sequence, and PCR is carried out by use of the respective primers to determine efficiencies of adenylation. Subsequently, an anchor sequence that is more likely to undergo adenylation is screened to decide an anchor sequence more likely undergo adenylation, followed by PCR by use of a primer having the decided anchor sequence.

    摘要翻译： 在PCR过程中DNA聚合酶的DNA片段的腺苷酸化发生，因此检测到两个峰。 为了防止峰分裂，需要提高腺苷酸化的效率，单峰发生而不改变反应条件。 为此，分别在5'末端具有锚定序列以使A，C，G或T中的任一个连接到锚定序列的5'末端的4种PCR引物，并进行PCR 通过使用各自的引物来确定腺苷酸化的效率。 随后，筛选更可能进行腺苷酸化的锚定序列以确定锚定序列更可能进行腺苷酸化，随后通过使用具有决定的锚定序列的引物进行PCR。

    Method for assaying DNA fragments in mixture
    4.
    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
    5.
    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。

    Method for determining nucleic acids base sequence and apparatus therefor
    6.
    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
    7.
    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
    8.
    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。

    Cell measuring and sorting chip
    10.
    发明授权
    Cell measuring and sorting chip 有权
    电池测量和分选芯片

    公开(公告)号：US08361412B2

    公开(公告)日：2013-01-29

    申请号：US11317004

    申请日：2005-12-27

    申请人： Kenji Yasuda ,  Akihiro Hattori ,  Kazunori Okano

    发明人： Kenji Yasuda ,  Akihiro Hattori ,  Kazunori Okano

    IPC分类号： B01L3/00

    CPC分类号： G01N15/147 ,  B01L3/502761 ,  B01L2200/0647 ,  B01L2300/0681 ,  B01L2300/0816 ,  B01L2300/0864 ,  B01L2400/0415 ,  B01L2400/0487 ,  C12M47/04 ,  G01N35/085 ,  G01N2015/149 ,  G06T7/0012 ,  G06T2207/10056 ,  G06T2207/30024

    摘要： A cell sorting device using an inexpensive chip capable of being exchanged for each sample. The chip includes: a first flow path allowing buffer fluid containing cells to flow down; second and third flow paths which put the first flow path therebetween and allow buffer fluid not containing cells to flow down; a fourth flow path which allows the buffer fluid as a single flow path formed by joining the buffer fluids in the other three flow paths; a cell detecting region for detecting cells flowing with the buffer fluid down the fourth flow path; and a cell sorting region for sorting the cells according to a type of the cells detected. The first to fourth flow paths are cascaded, are supplied with the buffer fluid from reservoirs with the same fluid level, and have substantially the same width or cross-section area.

    摘要翻译： 一种使用廉价芯片的细胞分选装置，能够对每个样品进行交换。 芯片包括：允许包含电池的缓冲流体向下流动的第一流动路径; 第二和第三流动路径，其中第一流动路径在其间并且允许不包含电池的缓冲流体向下流动; 第四流路，其允许缓冲流体作为通过连接其它三个流动路径中的缓冲流体形成的单个流动路径; 细胞检测区域，用于检测沿着第四流动路径流动的缓冲液的细胞; 以及用于根据检测到的细胞的类型对细胞进行分选的细胞分选区域。 第一至第四流动路径级联，从具有相同流体水平的储存器提供缓冲流体，并且具有基本上相同的宽度或横截面面积。