公开(公告)号：US06867027B1

公开(公告)日：2005-03-15

申请号：US09254344

申请日：1998-07-06

申请人： Yoshihide Hayashizaki ,  Masanori Watahiki

发明人： Yoshihide Hayashizaki ,  Masanori Watahiki

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

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聚合酶中的至少一个氨基酸被修饰以增强其与相应的野生型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，例如公开了其中存在于野生型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替代的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在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。 不同的基组。

公开(公告)号：US06544736B1

公开(公告)日：2003-04-08

申请号：US09508753

申请日：2000-06-16

申请人： Akira Shimamoto ,  Yasuhiro Furuichi ,  Yuko Shibata ,  Hiroko Funaki ,  Eiji Ohara ,  Masanori Watahiki

发明人： Akira Shimamoto ,  Yasuhiro Furuichi ,  Yuko Shibata ,  Hiroko Funaki ,  Eiji Ohara ,  Masanori Watahiki

IPC分类号： C12Q168

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'末端的磷酸基。 在第二步，除去mRNA样品中全长mRNA的5'末端的帽结构。 在第三步骤中，通过第一和第二步骤产生的mRNA的5'端的寡核糖核苷酸与磷酸基连接。 在第四步骤中，使用与mRNA内部的中间序列退火的短链寡核苷酸作为引物，将第三步的5'-末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。