公开(公告)号：EP4269592A1

公开(公告)日：2023-11-01

申请号：EP21911024.4

申请日：2021-12-24

申请人： Nippon Gene Co., Ltd

发明人： ASANO Shizuko ,  KOKUTANI Ryota ,  MINEGISHI Yasutaka ,  MAKI Fuminori ,  IZAWA Masaki ,  YONEDA Yuko

IPC分类号： C12N15/54 ,  C12M1/00 ,  C12N1/15 ,  C12N1/19 ,  C12N1/21 ,  C12N5/10 ,  C12N9/12 ,  C12N15/63 ,  C12Q1/6844

摘要： An object of the present invention is to provide a novel modified thermostable DNA polymerase having a reverse transcription activity. According to the present invention, there is provided a DNA polymerase having an activity to catalyze a reverse transcription reaction in the presence of Mg 2+ and comprising an amino acid sequence having an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 5 and amino acid substitutions at both positions 744 and 745 with positively charged amino acids.

公开(公告)号：EP3012326A1

公开(公告)日：2016-04-27

申请号：EP14775157.2

申请日：2014-03-26

申请人： Nippon Gene Co., Ltd

发明人： KITANI, Masakazu ,  MAKI, Fuminori ,  IZAWA, Masaki ,  KANAYAMA, Shinji ,  YONEDA, Yuko

IPC分类号： C12Q1/68 ,  C12N15/09

CPC分类号： C12Q1/6827 ,  C12Q1/6806 ,  C12Q1/6818 ,  C12Q1/6853 ,  C12Q2525/155 ,  C12Q2525/301 ,  C12Q2531/119 ,  C12Q2565/101

摘要： By establishing a simple method for extracting a nucleic acid from a human specimen, and using the LAMP method, which is an isothermal gene amplification method showing superior quickness and convenience, with a concept of measurement different from the conventional ones, there is established a test method including steps up to detection with a measurement apparatus. There is provided a method for detecting a target site, which is an LAMP method using four kinds of primers, FIP, F3 primer, BIP, and B3 primer, which are designed on the basis of six regions of a template polynucleotide, F1 region, F2 region, F3 region, B1 region, B2 region, and B3 region, wherein: the template polynucleotide contains the target site, the four kinds of primers are designed so that the target site exists between the F1 region and the F2 region, or between the B1 region and the B2 region; a loop primer designed on the basis of a region between the F 1 region and the F2 region or between the B 1 region and the B2 region on the side on which the target site exists, and an oligonucleotide probe that can associate with a region including the target site are used, the loop primer and the probe are designed so that the 5' end of the loop primer and the 3' end of the probe associate with the template polynucleotide at positions close to each other, one of the loop primer and the probe is modified with a fluorescent molecule around the 5' end in the case of the loop primer or the 3' end in the case of the probe, and the other is modified with a quenching molecule.

摘要翻译： 通过建立从人体标本提取核酸的简单方法，使用作为具有优异的快速性和便利性的等温基因扩增方法的LAMP方法，与常规测量方法不同的测量概念， 方法包括直到用测量装置检测的步骤。 提供了用于检测靶位点的方法，该方法是使用四种引物，FIP，F3引物，BIP和B3引物的LAMP方法，其基于模板多核苷酸的F1区， F2区域，F3区域，B1区域，B2区域和B3区域，其中：模板多核苷酸含有靶位点，设计四种引物以使得目标位点存在于F1区域和F2区域之间，或者在 B1区域和B2区域; 基于F 1区域和F2区域之间的区域或者在目标部位存在的一侧的B 1区域和B2区域之间的区域设计的环状引物，以及可以与包含 使用靶位点，设计环引物和探针，使得环引物的5'末端和探针的3'端与模板多核苷酸在彼此接近的位置相关联，其中一个环引物和 在环状引物的情况下，探针在5'末端附近的荧光分子被修饰，在探针的情况下用3'端进行修饰，另外用猝灭分子进行修饰。

公开(公告)号：EP3012326A4

公开(公告)日：2016-12-21

申请号：EP14775157

申请日：2014-03-26

申请人： NIPPON GENE CO LTD

发明人： KITANI MASAKAZU ,  MAKI FUMINORI ,  IZAWA MASAKI ,  KANAYAMA SHINJI ,  YONEDA YUKO

IPC分类号： C12Q1/68 ,  C12N15/09

CPC分类号： C12Q1/6827 ,  C12Q1/6806 ,  C12Q1/6818 ,  C12Q1/6853 ,  C12Q2525/155 ,  C12Q2525/301 ,  C12Q2531/119 ,  C12Q2565/101

摘要： By establishing a simple method for extracting a nucleic acid from a human specimen, and using the LAMP method, which is an isothermal gene amplification method showing superior quickness and convenience, with a concept of measurement different from the conventional ones, there is established a test method including steps up to detection with a measurement apparatus. There is provided a method for detecting a target site, which is an LAMP method using four kinds of primers, FIP, F3 primer, BIP, and B3 primer, which are designed on the basis of six regions of a template polynucleotide, F1 region, F2 region, F3 region, B1 region, B2 region, and B3 region, wherein: the template polynucleotide contains the target site, the four kinds of primers are designed so that the target site exists between the F1 region and the F2 region, or between the B1 region and the B2 region; a loop primer designed on the basis of a region between the F 1 region and the F2 region or between the B 1 region and the B2 region on the side on which the target site exists, and an oligonucleotide probe that can associate with a region including the target site are used, the loop primer and the probe are designed so that the 5' end of the loop primer and the 3' end of the probe associate with the template polynucleotide at positions close to each other, one of the loop primer and the probe is modified with a fluorescent molecule around the 5' end in the case of the loop primer or the 3' end in the case of the probe, and the other is modified with a quenching molecule.

公开(公告)号：EP1016729A4

公开(公告)日：2003-05-14

申请号：EP98943012

申请日：1998-09-17

申请人： NIPPON GENE CO LTD ,  AGENE RES INST CO LTD

发明人： SHIMAMOTO AKIRA ,  FURUICHI YASUHIRO ,  SHIBATA YUKO ,  FUNAKI HIROKO ,  OHARA EIJI ,  WATAHIKI MASANORI

IPC分类号： C12N15/10 ,  C12Q1/68 ,  C12N9/16 ,  C12N15/11

CPC分类号： C12N15/1096

摘要： A method for synthesizing a cDNA containing the 5'-terminal sequence of a full-length mRNA having a cap structure from an mRNA sample containing the full-length mRNA having the above cap structure together with a non-full-length mRNA having no cap structure. This method comprises the first step of eliminating the 5'-terminal phosphate group of the non-full-length mRNA in the mRNA sample, the second step of eliminating the 5'-terminal cap structure of the full-length mRNA in the mRNA sample, the third step of bonding an oligonucleotide to the 5'-terminal phosphate group of the mRNA which has been formed in the sample via the first and second steps, and the fourth step of synthesizing the first strand cDNA by subjecting to a treatment with a reverse transcriptase the mRNA carrying the oligonucleotide bonded to the 5'-terminal phosphate group thereof in the third step with the use of a primer comprising a short-chain oligonucleotide annealing with a sequence in the middle of the mRNA.

公开(公告)号：EP0939130A4

公开(公告)日：2002-09-18

申请号：EP98929851

申请日：1998-07-06

申请人： RIKAGAKU KENKYUSHO ,  NIPPON GENE CO LTD ,  HAYASHIZAKI YOSHIHIDE

发明人： HAYASHIZAKI YOSHIHIDE ,  WATAHIKI MASANORI

IPC分类号： C12N15/09 ,  C12N1/21 ,  C12N9/12 ,  C12N15/54 ,  C12Q1/68 ,  C12R1/19 ,  C12P19/34

CPC分类号： C12N9/1247 ,  C12N9/1252

摘要： An RNA polymerase comprising a wild type RNA polymerase with at least one amino acid modified so as to have a higher ability of incorporating 3'-deoxyribonucleotide or a derivative thereof than that of the corresponding wild type RNA polymerase. Specifically, for example, an RNA polymerase comprising a wild type RNA polymerase wherein at least one amino acid present in the nucleotide bonding site, for example, phenylalanine, is substituted by tyrosine. The RNA polymerase has little or no bias of incorporation between ribonucleotide and 3'-deoxyribonucleotide, between ribonucleotides having different bases, and between deoxyribonucleotides having different bases.

公开(公告)号：EP0939130A1

公开(公告)日：1999-09-01

申请号：EP98929851.8

申请日：1998-07-06

申请人： THE INSTITUTE OF PHYSICAL & CHEMICAL RESEARCH ,  Nippon Gene Co., Ltd ,  Hayashizaki, Yoshihide

发明人： HAYASHIZAKI, Yoshihide, Inst. Phys. and Chem. Res. ,  WATAHIKI, Masanori, 25-13, Kamikunishige

IPC分类号： C12N15/54 ,  C12N9/12 ,  C12N1/21 ,  C12Q1/68 ,  C12P19/34

CPC分类号： C12N9/1247 ,  C12N9/1252

摘要： Disclosed are RNA polymerases consisting of a wild type RNA polymerase provided that at least one of amino acids in the wild type RNA polymerase has been modified to enhance its ability for incorporating 3'-deoxyribonucleotides and derivatives thereof in comparison with the corresponding wild type RNA polymerases. Specifically, disclosed are, for example, the RNA polymerases wherein at least one amino acid present in a nucleotide binding sites of the wild type RNA polymerases such as phenylalanine has been replaced with tyrosine. The RNA polymerases of the present invention are a RNA polymerase which exhibits little or no bias for incorporation between ribonucleotides and 3'-deoxyribonucleotide as well as among ribonucleotides having different base groups and among deoxyribonucleotides having different base groups.

摘要翻译： 公开了由野生型RNA聚合酶组成的RNA聚合酶，只要野生型RNA聚合酶中的至少一种氨基酸已被修饰以增强其与相应野生型RNA聚合酶相比的3'-脱氧核糖核苷酸及其衍生物的结合能力 。 具体地，例如公开了存在于野生型RNA聚合酶的核苷酸结合位点如苯丙氨酸中的至少一个氨基酸已被酪氨酸取代的RNA聚合酶。 本发明的RNA聚合酶是对于核糖核苷酸和3'-脱氧核糖核苷酸之间以及具有不同碱基的核糖核苷酸和具有不同碱基的脱氧核糖核苷酸之间并入显示很少或没有偏差的RNA聚合酶。

公开(公告)号：EP0939130B1

公开(公告)日：2006-01-04

申请号：EP98929851.8

申请日：1998-07-06

申请人： THE INSTITUTE OF PHYSICAL & CHEMICAL RESEARCH ,  Nippon Gene Co., Ltd ,  Nippon Genetech Co. Ltd.

发明人： HAYASHIZAKI, Yoshihide, Inst. Phys. and Chem. Res. ,  WATAHIKI, Masanori

IPC分类号： C12N15/54 ,  C12N9/12 ,  C12N1/21 ,  C12Q1/68 ,  C12P19/34 ,  C12R1/19

CPC分类号： C12N9/1247 ,  C12N9/1252

摘要： An RNA polymerase comprising a wild type RNA polymerase with at least one amino acid modified so as to have a higher ability of incorporating 3'-deoxyribonucleotide or a derivative thereof than that of the corresponding wild type RNA polymerase. Specifically, for example, an RNA polymerase comprising a wild type RNA polymerase wherein at least one amino acid present in the nucleotide bonding site, for example, phenylalanine, is substituted by tyrosine. The RNA polymerase has little or no bias of incorporation between ribonucleotide and 3'-deoxyribonucleotide, between ribonucleotides having different bases, and between deoxyribonucleotides having different bases.

公开(公告)号：EP1016729A1

公开(公告)日：2000-07-05

申请号：EP98943012.9

申请日：1998-09-17

申请人： Nippon Gene Co., Ltd ,  Agene Research Institute, Co., Ltd.

发明人： SHIMAMOTO, Akira ,  FURUICHI, Yasuhiro ,  SHIBATA, Yuko ,  FUNAKI, Hiroko ,  OHARA, Eiji ,  WATAHIKI, Masanori

IPC分类号： C12Q1/68 ,  C12N15/11 ,  C12N9/16

CPC分类号： C12N15/1096

摘要： cDNA including the 5'-terminal sequence of full-length mRNA with a cap structure is synthesized from a mRNA sample containing the full-length mRNA with the cap structure and non-full-length mRNA without any cap structure in mixture. At the first step, the phosphate group at 5'-terminus of the non-full-length mRNA in the mRNA sample is removed. At the second step, the cap structure at the 5'-terminus of the full-length mRNA in the mRNA sample is removed. At the third step, an oligoribonucleotide is ligated to the phosphate group at 5'-terminus of mRNA generated through the first and second steps. At the fourth step, mRNA with the oligoribonucleotide ligated at the 5'-terminus thereof at the third step is subjected to a reverse transcriptase process using a short-chain oligonucleotide capable of being annealed to an intermediate sequence within the mRNA as primer, to synthesize a first-strand cDNA.

摘要翻译： 包含具有帽结构的全长mRNA的5'末端序列的cDNA由含有具有帽结构的全长mRNA的mRNA样品和不具有任何盖结构的非全长mRNA合成。 在第一步，除去mRNA样品中非全长mRNA的5'端的磷酸基。 在第二步，除去mRNA样品中全长mRNA的5'端的帽结构。 在第三步骤中，通过第一和第二步产生的mRNA的5'末端的寡核苷酸与磷酸基连接。 在第四步骤中，使用能够与mRNA内部的中间序列退火的短链寡核苷酸作为引物，在第三步骤的将其在其5'端连接的寡核糖核苷酸的mRNA进行逆转录酶处理，合成 第一链cDNA。