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    • 10. 发明申请
    • PROCESS OF CLEAN CLONING
    • 清洁过程
    • WO2010040531A3
    • 2010-07-01
    • PCT/EP2009007237
    • 2009-10-08
    • ICON GENETICS GMBHMARILLONNET SYLVESTREWERNER STEFANENGLER CAROLAKANDZIA ROMYTHIEME FRANKWEBER ERNST
    • MARILLONNET SYLVESTREWERNER STEFANENGLER CAROLAKANDZIA ROMYTHIEME FRANKWEBER ERNST
    • C12N15/09C12N15/10
    • C12N15/64C12N15/66
    • A process of inserting a nucleic acid sequence of interest into an acceptor nucleic acid, comprising the following steps: amplifying by PCR a DNA comprising in the following order a sequence segment U, a nucleic acid sequence segment of known nucleotide sequence K2 and a nucleic acid sequence segment of known sequence K3 using a forward primer defining a first end of the amplified DNA and a reverse primer defining a second end of the amplified DNA, said reverse primer terminating at its 3'-end in a nucleotide sequence of nucleic acid sequence segment K3; treating the linear double-stranded DNA molecules contained in the PCR product obtained in the previous step with an exonuclease to obtain a single-stranded overhang at the first end of the DNA and a single-stranded overhang comprising nucleic acid segments K2 and K3 at the second end of the DNA; and annealing the product of the previous step to a linearized double-stranded acceptor nucleic acid having at a first end thereof a single-stranded overhang complementary to the single-stranded overhang of the first end of the DNA and at a second end thereof a single- stranded overhang complementary to the single-stranded sequence segment K2 of the second end of the DNA.
    • 包括以下步骤:通过PCR扩增包含以下顺序的DNA序列的已知核苷酸序列K2的核酸序列片段和核酸的方法 已知序列K3的序列片段使用限定扩增DNA第一末端的正向引物和限定扩增DNA第二末端的反向引物,所述反向引物在其核酸序列片段的核苷酸序列的3'末端终止 K3; 用外切核酸酶处理前一步骤中获得的PCR产物中包含的线性双链DNA分子,以在DNA的第一端获得单链突出端,并且在第一端包含核酸片段K2和K3的单链突出端 DNA的第二端; 并将前述步骤的产物退火到线性双链受体核酸,其在第一端具有与DNA的第一末端的单链突出部互补的单链突出端,并且在其第二端具有单链突出端 - 与DNA的第二末端的单链序列片段K2互补的双链突出端。