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    • 64. 发明授权
    • Plasmid
    • 质粒
    • US6165749A
    • 2000-12-26
    • US952089
    • 1997-11-10
    • Hiroaki SagawaHarumi UenoAtsushi OshimaIkunoshin Kato
    • Hiroaki SagawaHarumi UenoAtsushi OshimaIkunoshin Kato
    • C12N15/09C12N1/21C12N9/10C12N9/16C12N9/22C12N15/55C12N15/69C12R1/19C12P21/06
    • C12N15/69C12N9/1007C12N9/22
    • A plasmid vector characterized by comprising a promoter sequence that can be recognized by an RNA polymerase which is not inherent in a host and that controls the expression of desired genes and a replication origin that increases the number of copies under the induction by exogenous factors; methods for expression and isolation of target genes by using the vector; a polypeptide having the activity of an AccIII restriction endonuclease; and a DNA encoding the polypeptide. The invention provides for the first time a plasmid vector which can introduce an exogenous desired gene encoding proteins which are lethal or harmful to hosts into the hosts, a method for efficiently expressing the proteins by using the vector, and also a method for permitting a restriction endonuclease gene constituting a restriction-modification system to be isolated even in the absence of a modification enzyme gene, which has been difficult in the prior arts.
    • PCT No.PCT / JP97 / 00748 Sec。 371日期:1997年11月10日 102(e)日期1997年11月10日PCT 1997年3月10日PCT公布。 第WO97 / 34006号公报 日期1997年9月18日一种质粒载体,其特征在于包含可由RNA聚合酶识别的启动子序列,其不是宿主中固有的并且控制所需基因的表达和增加诱导下拷贝数的复制起点 由外生因素; 通过使用载体表达和分离靶基因的方法; 具有AccIII限制性内切核酸酶活性的多肽; 和编码该多肽的DNA。 本发明首次提供能够将编码对宿主具有致死性或有害性的蛋白质的外源性所需基因导入宿主的质粒载体,使用该载体有效表达蛋白质的方法,以及允许限制的方法 构成即使在不存在修饰酶基因的情况下分离的限制性修饰系统的核酸内切酶基因,这在现有技术中是困难的。
    • 68. 发明授权
    • Method for synthesizing DNA
    • DNA合成方法
    • US06673578B1
    • 2004-01-06
    • US09786684
    • 2001-05-22
    • Takashi UemoriYoshimi SatoMariko OkawaTomoko FujitaKazue MiyakeOsamu TakedaHiroaki SagawaMichio HagiyaHiroyuki MukaiKiyozo AsadaIkunoshin Kato
    • Takashi UemoriYoshimi SatoMariko OkawaTomoko FujitaKazue MiyakeOsamu TakedaHiroaki SagawaMichio HagiyaHiroyuki MukaiKiyozo AsadaIkunoshin Kato
    • C12P1934
    • C12N15/1003C12Q1/686C12Q2527/125
    • A DNA synthesis method with a shortened time period required for DNA synthesis by polymerase chain reaction (PCR), characterized in that a DNA polymerase is used in an amount effective for providing more than 10 ng of amplified DNA fragments of about 2 kb per 50 &mgr;l of a reaction mixture, when PCR is carried out under the following conditions (A) and (B): (A) reaction mixture: 50 &mgr;l volume of a reaction mixture comprising DNA polymerase, 1 ng of genomic DNA from Escherichia coli, and 10 pmol each of primers Eco-1 and Eco-2 (nucleotide sequences of the primers Eco-1 and Eco-2 being shown in SEQ ID NOs: 10 and 11 of Sequence Listing, respectively); and having a composition suitable for the DNA polymerase; and (B) reaction conditions: 35 cycles of PCR, wherein one cycle consists of 99° C., 1 second-66° C., 7 seconds; a kit for DNA synthesis usable for the DNA synthesis method; and an article of manufacture of a PCR agent. According to the present invention, the procedures in the genetic engineering studies and industries involved with PCR can be speeded up.
    • 一种DNA合成方法,其通过聚合酶链式反应(PCR)进行DNA合成所需的时间缩短,其特征在于使用DNA聚合酶,其量有效提供超过10ng每50ul约2kb的扩增DNA片段 的反应混合物,当在以下条件(A)和(B)下进行PCR时:(A)反应混合物:将50体积的包含DNA聚合酶的反应混合物,1ng来自大肠杆菌的基因组DNA和10ng 各引物Eco-1和Eco-2(引物Eco-1和Eco-2的核苷酸序列分别示于序列表的SEQ ID NO:10和11中) 并具有适合于DNA聚合酶的组合物; 和(B)反应条件:35个循环的PCR,其中一个循环由99℃,1秒-66℃,7秒; 用于DNA合成方法的DNA合成试剂盒; 以及PCR试剂的制造。 根据本发明,可以加快基因工程研究和涉及PCR的行业的程序。