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1. 发明申请

US20050260631A1 Method of amplifying nucleic acids, reagent kit for amplifying nucleic acids, method of detecting single nucleotide polymorphism, and reagent kit for detecting single nucleotide polymorphism 审中-公开
标题翻译：核酸扩增方法，核酸扩增试剂盒，单核苷酸多态性检测方法，单核苷酸多态性检测试剂盒
公开(公告)号：US20050260631A1
公开(公告)日：2005-11-24
申请号：US11081949
申请日：2005-03-17
申请人： Yasushi Shigemori , Takehiko Shibata , Tsutomu Mikawa , Michio Oishi , Osamu Ohara
发明人： Yasushi Shigemori , Takehiko Shibata , Tsutomu Mikawa , Michio Oishi , Osamu Ohara
IPC分类号： C12N15/09 , C12Q1/68 , C12P19/34
CPC分类号： C12Q1/6848 , C12Q1/6858 , C12Q2521/507
摘要： An object of the present invention is to provide a nucleic acid amplification method for amplifying a desired nucleic acid while suppressing amplification of byproducts in a PCR reaction, a reagent kit used for nucleic acid amplification, a method of detecting single nucleotide polymorphism to detect single nucleotide polymorphism by utilizing that amplification of byproducts is suppressed in a PCR reaction, and a reagent kit used for detecting single nucleotide polymorphism. The method of amplifying nucleic acids by PCR is characterized by admixing in a reaction solution, a homologous recombinant protein which contains at least one of a RecA protein derived from Thermus thermophilus, and a modified RecA protein obtained by modification of the RecA protein and having a function similar to that of the RecA protein, and carrying out PCR.
摘要翻译：本发明的目的是提供用于扩增所需核酸同时抑制PCR反应中副产物扩增的核酸扩增方法，用于核酸扩增的试剂盒，检测单核苷酸多态性以检测单核苷酸的方法在PCR反应中抑制副产物扩增的多态性，以及用于检测单核苷酸多态性的试剂盒。通过PCR扩增核酸的方法的特征在于在反应溶液中混合含有至少一种源自Thermus thermophilus的RecA蛋白的同源重组蛋白，以及通过修饰RecA蛋白获得的经修饰的RecA蛋白，并且具有功能类似于RecA蛋白，并进行PCR。

2. 发明授权

US07022501B1 Ligation of double-stranded DNAs 失效
标题翻译：双链DNA的连接
公开(公告)号：US07022501B1
公开(公告)日：2006-04-04
申请号：US09607361
申请日：2000-06-30
申请人： Yasushi Shigemori , Michio Oishi
发明人： Yasushi Shigemori , Michio Oishi
IPC分类号： C12P19/34 , C12P21/06 , C12N15/64 , C12N9/00 , C22N1/20
CPC分类号： C12N15/10
摘要： The present invention relates to a DNA ligation method. Specifically, a DNA complex comprising a three-stranded structure in each of the ends of double-stranded DNAs, one of which has single-stranded regions at the end, and the other of which does not have single-stranded regions at the end, is formed under the presence of a homologous recombinant protein. Moreover, according to needs, the DNA complex is introduced into host cells, and said cells are cultured.
摘要翻译：本发明涉及DNA连接方法。具体而言，在双链DNA的各末端具有三链结构的DNA复合体，其中一个末端具有单链区域，另一个在末端没有单链区域，在同源重组蛋白的存在下形成。此外，根据需要，将DNA复合物引入宿主细胞，并培养所述细胞。

3. 发明授权

US07713700B2 Method of amplifying nucleic acids, reagent kit for amplifying nucleic acids, method of detecting single nucleotide polymorphism, and reagent kit for detecting single nucleotide polymorphism 失效
标题翻译：核酸扩增方法，核酸扩增试剂盒，单核苷酸多态性检测方法，单核苷酸多态性检测试剂盒
公开(公告)号：US07713700B2
公开(公告)日：2010-05-11
申请号：US12062215
申请日：2008-04-03
申请人： Yasushi Shigemori , Takehiko Shibata , Tsutomu Mikawa , Michio Oishi , Osamu Ohara
发明人： Yasushi Shigemori , Takehiko Shibata , Tsutomu Mikawa , Michio Oishi , Osamu Ohara
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6848 , C12Q1/6858 , C12Q2521/507
摘要： An object of the present invention is to provide a nucleic acid amplification method for amplifying a desired nucleic acid while suppressing amplification of byproducts in a PCR reaction, a reagent kit used for nucleic acid amplification, a method of detecting single nucleotide polymorphism to detect single nucleotide polymorphism by utilizing that amplification of byproducts is suppressed in a PCR reaction, and a reagent kit used for detecting single nucleotide polymorphism. The method of amplifying nucleic acids by PCR is characterized by admixing in a reaction solution, a homologous recombinant protein which contains at least one of a RecA protein derived from Thermus thermophilus, and a modified RecA protein obtained by modification of the RecA protein and having a function similar to that of the RecA protein, and carrying out PCR.
摘要翻译：本发明的目的是提供用于扩增所需核酸同时抑制PCR反应中副产物扩增的核酸扩增方法，用于核酸扩增的试剂盒，检测单核苷酸多态性以检测单核苷酸的方法在PCR反应中抑制副产物扩增的多态性，以及用于检测单核苷酸多态性的试剂盒。通过PCR扩增核酸的方法的特征在于在反应溶液中混合含有至少一种源自Thermus thermophilus的RecA蛋白的同源重组蛋白，以及通过修饰RecA蛋白获得的经修饰的RecA蛋白，并且具有功能类似于RecA蛋白，并进行PCR。

4. 发明授权

US06541226B1 Method for specifically cleaving double-stranded DNA and kit for the method 失效
标题翻译：用于特异性切割双链DNA的方法和该方法的试剂盒
公开(公告)号：US06541226B1
公开(公告)日：2003-04-01
申请号：US09549949
申请日：2000-04-14
申请人： Yasushi Shigemori , Michio Oishi
发明人： Yasushi Shigemori , Michio Oishi
IPC分类号： C12N1564
CPC分类号： C12N15/113 , C12N2310/15 , C12N2310/153 , C12Q1/6839 , C12Y301/30001 , C12Q2527/125 , C12Q2521/507 , C12Q2521/307
摘要： A method for specifically cleaving a double-stranded DNA. The method comprises forming a three-stranded portion comprising the double-stranded DNA portion including the position to be cleaved or its vicinity and an oligonucleotide and treating the three-stranded protion with a nuclease.
摘要翻译：一种用于特异性切割双链DNA的方法。该方法包括形成包含待切割位置或其附近的双链DNA部分的三链部分和寡核苷酸，并用核酸酶处理三链引物。

5. 发明授权

US06849410B2 Method for detecting DNA polymorphism applying triple strand DNA formation technique 失效
标题翻译：检测应用三链DNA形成技术的DNA多态性的方法
公开(公告)号：US06849410B2
公开(公告)日：2005-02-01
申请号：US09989526
申请日：2001-11-21
申请人： Yasushi Shigemori , Michio Oishi , Osamu Ohara
发明人： Yasushi Shigemori , Michio Oishi , Osamu Ohara
IPC分类号： C12N15/09 , C07H21/02 , C07H21/04 , C12P19/34 , C12Q1/68
CPC分类号： C12Q1/6827 , C12Q1/6839 , C12Q2537/119 , C12Q2527/107 , C12Q2521/507
摘要： An objective of this invention is to provide a method for detecting DNA polymorphism that has high sensitivity and efficiency and does not need long DNA searching region.A homologous recombination protein RecA makes partial triple strand DNA from target double DNA and oligonucleotide probe complementary to the DNA. The triple strand DNA maintains stable triple strand DNA after RecA protein is removed. The present inventors found that the thermostability of triple strand DNA changes greatly when there is a mismatch between target DNA and oligonucleotide probe because of the existence of polymorphism in the target DNA. Utilizing this change of thermostability, efficient detection of polymorphism in labeled DNA is possible by examining whether oligonucleotide probe is released and the triple strand DNA is solved after heat treatment of triple strand DNA formed using homologous recombination protein.
摘要翻译：本发明的目的是提供一种检测DNA多态性的方法，其具有高灵敏度和高效性并且不需要长的DNA搜索区。同源重组蛋白RecA使得来自靶双重DNA的部分三链DNA和与DNA互补的寡核苷酸探针。在除去RecA蛋白后，三链DNA保持稳定的三链DNA。本发明人发现，由于目标DNA中存在多态性，当靶DNA和寡核苷酸探针之间存在错配时，三链DNA的热稳定性变化很大。利用这种热稳定性的变化，可以通过检查寡核苷酸探针是否被释放并且在使用同源重组蛋白形成的三链DNA热处理后求解三链DNA来有效地检测标记的DNA中的多态性。

6. 发明申请

US20060160115A1 Activation method of protein derived from extremely thermophilic bacterium in nucleic acid amplification reaction and use thereof 有权
标题翻译：核酸扩增反应中极嗜温细菌衍生的蛋白质的活化方法及其应用
公开(公告)号：US20060160115A1
公开(公告)日：2006-07-20
申请号：US11298875
申请日：2005-12-12
申请人： Yasushi Shigemori
发明人： Yasushi Shigemori
IPC分类号： C12Q1/68 , C12P19/34 , C12N9/22
CPC分类号： C12Q1/6844 , C12Q1/686 , C12Q2521/107 , C12Q2522/101 , C12Q2527/127
摘要： The present invention is to provide a method which makes it possible to activate RecA, and maintain its biological function in all the PCR cycles. Also, the invention is to provide a nucleic acid amplification method, and a kit for amplifying a nucleic acid which makes it possible to suppress non-specific amplification more specifically and efficiently to amplify only the desired nucleic acid. A method of activating a RecA protein derived from an extremely thermophilic bacterium in a polymerase chain reaction carried out in the presence of the RecA protein derived from the extremely thermophilic bacterium, wherein the RecA protein is activated by carrying out the reaction with the addition of nucleotide 5′-triphosphate (provided that the nucleotide 5′-triphosphate is neither deoxynucleotide 5′-triphosphate nor nucleotide 5′-O-3-thiotriphosphate).
摘要翻译：本发明提供一种能够激活RecA并在所有PCR循环中维持其生物学功能的方法。此外，本发明提供核酸扩增方法和用于扩增核酸的试剂盒，其使得可以更具体地和有效地抑制非特异性扩增来扩增仅需要的核酸的试剂盒。在来自极嗜温细菌的RecA蛋白存在下，在聚合酶链反应中活化来自极嗜温细菌的RecA蛋白质的方法，其中通过加入核苷酸进行反应来活化RecA蛋白质 5'-三磷酸（假定核苷酸5'-三磷酸既不是脱氧核苷酸5'-三磷酸，也不是核苷酸5'-O-3-硫代三磷酸）。

7. 发明授权

US07902335B1 Heat-stable recA mutant protein and a nucleic acid amplification method using the heat-stable recA mutant protein 有权
标题翻译：热稳定recA突变蛋白和使用热稳定recA突变蛋白的核酸扩增方法
公开(公告)号：US07902335B1
公开(公告)日：2011-03-08
申请号：US11780284
申请日：2007-07-19
申请人： Yasushi Shigemori , Takehiko Shibata , Tsutomu Mikawa
发明人： Yasushi Shigemori , Takehiko Shibata , Tsutomu Mikawa
IPC分类号： C07K14/00 , C07H21/02 , C12P21/04
CPC分类号： C12N9/00 , C12Q1/6844 , C12Q2521/507
摘要： A heat-stable RecA mutant protein is obtained by mutation involving either deletion or substitution of at least one amino acid in an amino acid sequence composing an acid region at C-terminal end of a wild type heat-stable RecA protein, and has an improved ability, compared to the wild type heat-stable RecA protein, for contributing to an increase in an amplification specificity of a template nucleic acid in a nucleic acid amplification reaction.
摘要翻译：通过涉及构成野生型热稳定性RecA蛋白C末端的酸性区域的氨基酸序列中的至少一个氨基酸的缺失或取代的突变获得热稳定性RecA突变蛋白，并且具有改良与野生型热稳定RecA蛋白相比，有助于增加模板核酸在核酸扩增反应中的扩增特异性。

8. 发明申请

US20050136443A1 Nucleic acid amplification method 审中-公开
标题翻译：核酸扩增法
公开(公告)号：US20050136443A1
公开(公告)日：2005-06-23
申请号：US10948193
申请日：2004-09-24
申请人： Yasushi Shigemori
发明人： Yasushi Shigemori
IPC分类号： C12N15/09 , C12Q1/68 , C12P19/34
CPC分类号： C12Q1/686 , C12Q2521/507 , C12Q2522/101
摘要： A DNA amplification method of amplifying DNA by PCR comprises a process of preparing a mixed solution for PCR reaction by mixing a template DNA, a primer DNA, a RecA protein derived from Thermus thermophilus (T. th. RecA protein) and a phage T4 gene 32 protein, and a process of amplifying DNA by subjecting the prepared mixed solution for PCR reaction to PCR reaction.
摘要翻译：通过PCR扩增DNA的DNA扩增方法包括通过混合模板DNA，引物DNA，来源于嗜热栖热菌（T.th.RacA蛋白）的RecA蛋白和噬菌体T4基因来制备用于PCR反应的混合溶液的方法 32蛋白质，以及通过将制备的混合溶液进行PCR反应进行PCR反应来扩增DNA的方法。

9. 发明授权

US07973131B2 Extreme thermophile single-stranded DNA binding mutant protein, and nucleic acid isothermal amplification method of use thereof 有权
标题翻译：极性嗜热单链DNA结合突变蛋白，以及其使用的核酸等温扩增方法
公开(公告)号：US07973131B2
公开(公告)日：2011-07-05
申请号：US11495640
申请日：2006-07-31
申请人： Yasushi Shigemori , Takehiko Shibata , Tsutomu Mikawa
发明人： Yasushi Shigemori , Takehiko Shibata , Tsutomu Mikawa
IPC分类号： C07K1/00 , C07K14/00
CPC分类号： C07K14/195
摘要： The invention establishes a technology that allows non-specific amplification to be inhibited during nucleic acid amplification in an isothermal amplification reaction, such that the amplification efficiency is increased. The invention is a extreme thermophile single-stranded DNA binding mutant protein, having an amino acid sequence that expresses a function that can contribute to increasing an amplification efficiency of a template nucleic acid in an isothermal amplification reaction system that uses a strand displacement polymerase, and having in its amino acid sequence a mutation site where a mutation involving at least one of deletion, substitution, addition, and insertion of one or more amino acids in amino acid sequence of extreme thermophile single-stranded DNA binding protein has occurred, and a method of use thereof.
摘要翻译：本发明建立了在等温扩增反应的核酸扩增期间允许非特异性扩增被抑制的技术，使得扩增效率提高。本发明是一种极端嗜热单链DNA结合突变蛋白，其具有表达功能的氨基酸序列，该氨基酸序列可有助于增加使用链置换聚合酶的等温扩增反应系统中模板核酸的扩增效率，以及在其氨基酸序列中具有突变位点，其中发生了涉及极端嗜热性单链DNA结合蛋白的氨基酸序列中的一个或多个氨基酸的缺失，取代，添加和插入中的至少一个的突变，以及方法的使用。

10. 发明申请

US20060127978A1 Nucleic acid recombination method, host cell and expression vector 审中-公开
标题翻译：核酸重组方法，宿主细胞和表达载体
公开(公告)号：US20060127978A1
公开(公告)日：2006-06-15
申请号：US11071331
申请日：2005-03-04
申请人： Yasushi Shigemori , Takehiko Shibata , Tsutomu Mikawa
发明人： Yasushi Shigemori , Takehiko Shibata , Tsutomu Mikawa
IPC分类号： C12N15/09 , C12P21/06
CPC分类号： C12N15/902
摘要： This invention is to provide a nucleic acid recombination method which enables to carry out precisely homologous recombination of a desired target region, a host cell which enables to carry out precisely homologous recombination of a desired target region, and an expression vector which can be used for the nucleic acid recombination method and the host cell. The nucleic acid recombination method to carry out homologous recombination of a nucleic acid in a host cell employs a host cell which enables the expression and recombination of a RecA-like protein, and enables the expression of a RecX-like protein. And, the nucleic acid recombination method comprises a recombination step of a nucleic acid to carry out homologous recombination of the nucleic acid by the RecA-like protein expressed in the host cell, and a RecA inhibition step to inhibit the activity of the RecA-like protein by expressing the RecX-like protein.
摘要翻译：本发明提供一种核酸重组方法，其能够进行所需靶区域的精确同源重组，能够进行期望靶区域的精确同源重组的宿主细胞和可用于核酸重组方法和宿主细胞。用于在宿主细胞中进行核酸的同源重组的核酸重组方法使用能够表达和重组RecA样蛋白的宿主细胞，并且能够表达RecX样蛋白。并且，核酸重组方法包括核酸的重组步骤，以通过宿主细胞中表达的RecA样蛋白进行核酸的同源重组，以及抑制RecA样活性的RecA抑制步骤蛋白质通过表达RecX样蛋白。

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