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。