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    • 6. 发明授权
    • Process for producing cDNAs with complete length, process for producing
intermediates thereof and process for producing vectors containing
cDNAs with complete lengths
    • 用于生产具有完整长度的cDNA的方法,其制备中间体的方法和用于生产含有完整长度的cDNA的载体的方法
    • US5597713A
    • 1997-01-28
    • US244188
    • 1994-07-25
    • Seishi KatoShingo Sekine
    • Seishi KatoShingo Sekine
    • C12N15/09C12N15/10C12N15/66C12N15/11C12N15/64C12P19/34
    • C12N15/1096C12N15/66
    • A process for providing cDNAs containing full primary structure information of proteins by selectively synthesizing full-length cDNAs containing sequences starting from the capped region of mRNAs is disclosed. The invention provides a process for producing an intermediate for the synthesis of a full-length cDNA, which comprises the steps of treating mRNA extracted from cells to eliminate the phosphate group from the 5' end of an uncapped degraded mRNA; decapping from the 5' end of a capped intact mRNA; and ligating either a DNA oligonucleotide or a DNA-RNA chimeric oligonucleotide represented by the following general formula [I] to the 5' end phosphate group formed in the above step by the action of T4 RNA ligase, thereby selectively adding either the DNA oligonucleotide or the DNA-RNA chimeric oligonucleotide having an arbitrary sequence to the 5' end of the intact mRNA.5'-dN1-dN2- . . . -dNm-N1-N2- . . . -Nn-3' [I]The present invention also provides a process for synthesizing a full-length cDNA, which comprises linking a double-stranded DNA primer having a dT tail by annealing to a poly(A) tail of the 3' end of the intact mRNA having the DNA oligonucleotide or the DNA-RNA chimeric nucleotide added at the 5' end produced by the above-mentioned process, and then synthesizing the first strand cDNA complementary to the intact mRNA by a reverse transcriptase.
    • PCT No.PCT / JP93 / 01359 Sec。 371日期:1994年7月25日 102(e)日期1994年7月25日PCT提交1993年9月22日PCT公布。 第WO94 / 08001号公报 日期1994年04月14日公开了通过选择性地合成含有从mRNA封端区开始的序列的全长cDNA提供含有蛋白质全部一级结构信息的cDNA的方法。 本发明提供了一种制备用于合成全长cDNA的中间体的方法,其包括以下步骤:处理从细胞中提取的mRNA,以从未封端的降解的mRNA的5'末端消除磷酸基团; 从覆盖的完整mRNA的5'端开始切割; 并通过T4 RNA连接酶的作用将由以下通式[I]表示的DNA寡核苷酸或DNA-RNA嵌合寡核苷酸与上述步骤中形成的5'端磷酸基结合,从而选择性地添加DNA寡核苷酸或 具有与完整mRNA的5'末端任意序列的DNA-RNA嵌合寡核苷酸。 5'-dN1-dN2-。 。 。 -dNm-N1-N2-。 。 。 -Nn-3'[I]本发明还提供了一种合成全长cDNA的方法,其包括将具有dT尾的双链DNA引物与3'端的poly(A)末端退火连接 的具有通过上述方法制备的5'端加入的DNA寡核苷酸或DNA-RNA嵌合核苷酸的完整mRNA,然后通过逆转录酶合成与完整mRNA互补的第一链cDNA。