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1. 发明申请

US20070231808A1 Methods of nucleic acid analysis by single molecule detection 审中-公开
标题翻译：通过单分子检测进行核酸分析的方法
公开(公告)号：US20070231808A1
公开(公告)日：2007-10-04
申请号：US11441193
申请日：2006-05-26
申请人： Chifumi Gouda , Chihiro Uematsu , Takashi Anazawa , Masataka Shirai
发明人： Chifumi Gouda , Chihiro Uematsu , Takashi Anazawa , Masataka Shirai
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6858 , C12Q1/6818 , C12Q1/6827 , C12Q1/6862 , C12Q2531/125 , C12Q2525/301 , C12Q2521/501 , C12Q2565/107
摘要： This invention provides a method of nucleic acid analysis that enables highly accurate and sensitive quantitation by counting the number of molecules among a plurality of types of genes without amplifying specific genes and that enable reduction of quantitation limits. This method comprises steps of: allowing a polynucleotide comprising a first region having a sequence complementary to the target gene at the 3′ end, a second region having a sequence complementary to the target gene at the 5′ end, and a third region corresponding to a detection probe to hybridize to the target gene; allowing the 3′ end of the first region hybridized to the target gene to ligate to the 5′ end of the second region so as to obtain a circularized polynucleotide; with the use of the circularized polynucleotide as a template, performing a primer extension reaction using a primer having a sequence complementary to part of the circularized polynucleotide and a strand-displacement DNA polymerase; allowing a detection probe containing a sequence identical to the third region to hybridize to a sequence complementary to the third region that iteratively appears in a single-stranded portion of the extension product; and optically detecting the quantity of the detection probe hybridized to the extension product to thereby quantitate the target gene.
摘要翻译：本发明提供核酸分析方法，通过计数多种类型的基因中的分子数而不扩增特异性基因，并且能够降低定量限度，使得能够进行高度准确和灵敏的定量。该方法包括以下步骤：允许包含在3'末端具有与靶基因互补的序列的第一区域的多核苷酸，在5'端具有与靶基因互补的序列的第二区域和对应于与靶基因杂交的检测探针; 允许与靶基因杂交的第一区域的3'末端连接到第二区域的5'端，以获得环化多核苷酸; 使用环化多核苷酸作为模板，使用具有与部分环化多核苷酸互补序列的引物和链置换DNA聚合酶进行引物延伸反应; 允许包含与第三区域相同的序列的检测探针与与重复出现在延伸产物的单链部分中的第三区域互补的序列杂交; 并光学检测与延伸产物杂交的检测探针的量，从而定量靶基因。

2. 发明申请

US20070238125A1 Probe synthesis method for nucleic acid detection 审中-公开
标题翻译：探针合成方法用于核酸检测
公开(公告)号：US20070238125A1
公开(公告)日：2007-10-11
申请号：US11783124
申请日：2007-04-06
申请人： Chihiro Uematsu , Chifumi Gouda , Masataka Shirai , Takashi Anazawa
发明人： Chihiro Uematsu , Chifumi Gouda , Masataka Shirai , Takashi Anazawa
IPC分类号： C12Q1/68 , C12P19/34 , C12M3/00
CPC分类号： C12P19/34
摘要： The present invention relates to a probe synthesis method and a probe synthesis kit for nucleic acid detection, which are intended for the fluorophore labeling and detection of a target gene. In this method, a probe unit for analyte nucleic acid recognition having a base sequence specific to an analyte nucleic acid and plural labeled probe units each having a fluorophore labeled internal base are hybridized to oligonucleotides complementary thereto, and the adjacent probe units are bonded by ligation. As a result, an analytical probe with increased fluorescence intensity is synthesized easily and inexpensively, while the number of fluorophore labels is controlled.
摘要翻译：本发明涉及用于靶基因的荧光标记和检测的用于核酸检测的探针合成方法和探针合成试剂盒。在该方法中，具有分析物核酸特异性的碱基序列的分析物核酸识别用探针单元和各自具有荧光团标记的内部碱基的多个标记的探针单元与互补的寡核苷酸杂交，并且通过连接键合相邻的探针单元。结果，容易且廉价地合成了增加荧光强度的分析探针，同时控制了荧光标记的数量。

3. 发明申请

US20060197034A1 Counting system for flouescent molecules 有权
标题翻译：浮游分子计数系统
公开(公告)号：US20060197034A1
公开(公告)日：2006-09-07
申请号：US11349898
申请日：2006-02-09
申请人： Masataka Shirai , Tsuyoshi Sonehara , Takashi Anazawa , Chifumi Gouda , Chihiro Uematsu
发明人： Masataka Shirai , Tsuyoshi Sonehara , Takashi Anazawa , Chifumi Gouda , Chihiro Uematsu
IPC分类号： G01N21/64
CPC分类号： G01N21/6456 , G01N21/6428
摘要： Single molecule measurement is conducted by propagating a laser light under multiple reflection between two substrates constituting a flow channel for flowing a sample solution, exciting target molecules in the sample, detecting one-dimensional images of generated fluorescence by a one-dimensional detection portion, synthesizing two-dimensional images from the obtained one-dimensional images by a data synthesizing portion and measuring the concentration of target molecules in the sample solution by counting the number of target molecules based on the two-dimensional images, thereby measuring the concentration of the target molecules in the sample solution. These constitutions for single molecule measurement lead to high precision determination of a micro-amount of a biological material.
摘要翻译：通过在构成用于流动样品溶液的流动通道的两个基板之间的多个反射下传播激光来进行单分子测量，激发样品中的目标分子，通过一维检测部分检测产生的荧光的一维图像，合成通过数据合成部分来自所获得的一维图像的二维图像，并且通过基于二维图像计数目标分子的数量来测量样品溶液中的目标分子的浓度，由此测量目标分子的浓度在样品溶液中。用于单分子测量的这些结构导致生物材料微量的高精度测定。

4. 发明授权

US07439522B2 Counting system for fluorescent molecules 有权
标题翻译：荧光分子计数系统
公开(公告)号：US07439522B2
公开(公告)日：2008-10-21
申请号：US11349898
申请日：2006-02-09
申请人： Masataka Shirai , Tsuyoshi Sonehara , Takashi Anazawa , Chifumi Gouda , Chihiro Uematsu
发明人： Masataka Shirai , Tsuyoshi Sonehara , Takashi Anazawa , Chifumi Gouda , Chihiro Uematsu
IPC分类号： G01N21/64
CPC分类号： G01N21/6456 , G01N21/6428
摘要： Single molecule measurement is conducted by propagating a laser light under multiple reflection between two substrates constituting a flow channel for flowing a sample solution, exciting target molecules in the sample, detecting one-dimensional images of generated fluorescence by a one-dimensional detection portion, synthesizing two-dimensional images from the obtained one-dimensional images by a data synthesizing portion and measuring the concentration of target molecules in the sample solution by counting the number of target molecules based on the two-dimensional images, thereby measuring the concentration of the target molecules in the sample solution. These constitutions for single molecule measurement lead to high precision determination of a micro-amount of a biological material.
摘要翻译：通过在构成用于流动样品溶液的流动通道的两个基板之间的多个反射下传播激光来进行单分子测量，激发样品中的目标分子，通过一维检测部分检测产生的荧光的一维图像，合成通过数据合成部分来自所获得的一维图像的二维图像，并且通过基于二维图像计数目标分子的数量来测量样品溶液中的目标分子的浓度，由此测量目标分子的浓度在样品溶液中。用于单分子测量的这些结构导致生物材料微量的高精度测定。

5. 发明申请

US20070215817A1 Fluorescence detection system 审中-公开
标题翻译：荧光检测系统
公开(公告)号：US20070215817A1
公开(公告)日：2007-09-20
申请号：US11705446
申请日：2007-02-13
申请人： Masataka Shirai , Takashi Anazawa , Chihiro Uematsu , Chifumi Gouda
发明人： Masataka Shirai , Takashi Anazawa , Chihiro Uematsu , Chifumi Gouda
IPC分类号： G01N21/64
CPC分类号： G01N21/05 , G01J3/2803 , G01J3/4406 , G01N21/6428 , G01N21/6458 , G01N2021/0346
摘要： Single molecular measurement is conducted with a high throughput. Fluorescence from labeled target molecules flowing a sample flow cell is measured by a line CCD element to realize single molecular measurement with a high throughput. Where, the number of photo detecting pixels of the line CCD element is smaller than a value obtained by dividing an exposure time by pixel transfer rate.
摘要翻译：单分子测量以高产量进行。通过线CCD元件测量流过样品流通池的标记的靶分子的荧光，以实现高分辨率的单分子测量。其中，线CCD元件的光检测像素的数量比通过像素传送速率分割曝光时间所获得的值小。

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